Amide-based cationic lipids

ABSTRACT

The present invention provides novel amide-based cationic lipids of the general structure:                    
     or a salt, or solvate, or enantiomers thereof wherein; (a) Y is a direct link or an alkylene of 1 to about 20 carbon atoms; (b) R 1  is H or a lipophilic moiety; (c) R 2 , R 3 , and R 4  are positively charged moieties, or at least one but not all of R 2 , R 3 , or R 4  is a positive moiety and the remaining are independently selected from H, an alkyl moiety of 1 to about 6 carbon atoms, or a heterocyclic moiety of about 5 to about 10 carbon atoms; (d) n and p are independently selected integers from 0 to 8, such that the sum of n and o is from 1 to 16; (e) X −  is an anion or polyanion and (f) m is an integer from 0 to a number equivalent to the positive charge(s) present on the lipid; provided that if Y is a direct link and the sum of n and p is 1 then one of either R 3  or R 4  must have an alkyl moiety of at least 10 carbon atoms. 
     The present invention further provides compositions of these lipids with polyanionic macromolecules, methods for interfering with protein expression in a cell utilizing these compositions and a kit for preparing the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/327,392 filed Jun. 7, 1999 now U.S. Pat. No. 6,339,173, which is acontinuation of U.S. patent application Ser. No. 08/681,297, filed Jul.22, 1996, now U.S. Pat. No. 6,020,526 issued Feb. 1, 2000, which was acontinuation of U.S. patent application Ser. No. 08/505,802, filed Jul.21, 1995, now abandoned.

TECHNICAL FIELD

The present invention is directed to novel amide-based cationic lipidcompounds useful in lipid aggregates for the delivery of macromoleculesinto cells.

BACKGROUND OF THE INVENTION

Lipid aggregates, such as liposomes, have been previously reported to beuseful as agents for the delivery of macromolecules such as DNA, RNA,oligonucleotides, proteins, and pharmaceutical compounds into cells. Inparticular, lipid aggregates which include charged as well as unchargedlipids have been especially effective for delivering polyanionicmolecules to cells. The reported effectiveness of cationic lipids mayresult from charge inmteractions with cells which are said to bear a netnegative charge. It has also been postulated that the net positivecharge on the cationic lipid aggregates may enable them to bindpolyanions, such as nucleic acids. Lipid aggregates containing DNA havebeen reported to be effective agents for efficient transfection ofcells.

The structure of various types of lipid aggregates vary depending onfactors which include composition and methods of forming the aggregate.Lipid aggregates include, for example, liposomes, unilamellar vesicles,multilamellar vesicles, micelles and the like, and may have particlesizes in the nanometer to micrometer range. Various methods of makinglipid aggregates have been reported in the art. One type of lipidaggregate comprises phospholipid containing liposomes. An importantdrawback to the use of this type of aggregate as a cell delivery vehicleis that the liposome has a negative charge that reduces the efficiencyof binding to a negatively charged cell surface. It has been reportedthat positively charged liposomes that are able to bind DNA may beformed by combining cationic lipid compounds with phospholipids. Theseliposomes may then be utilized to transfer DNA into target cells. (See,e.g. Felgner et al., Proc. Nat. Acad. Sci. 84:7413-7417, 1987; Eppsteinet al. U.S. Pat. No. 4,897,355; Felgner et al. U.S. Pat. No. 5,264,618;and Gebeyehu et al. U.S. Pat. No. 5,334,761).

Known cationic lipids includeN[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium chloride (“DOTMA”)and combinations of DOTMA with dioleoylphosphatidylethanolamine (“DOPE”)are commercially available. Formulation of DOTMA, either by itself or in1:1 combination with DOPE, into liposomes by conventional techniques hasbeen reported. However, compositions comprising DOTMA have been reportedto show some toxicity to cells.

Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than ether,linkages to the propylamine. However, DOTAP is reported to be morereadily degraded by target cells. Other cationic lipids which representstructural modifications of DOTMA and DOTAP have also been reported.

Other reported cationic lipid compounds include those which have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) and dipalmitoyl-phosphatidylethanolamine 5-carboxyspermyl-amide(“DPPES”) (See, e.g. Behr et al., U.S. Pat. No. 5,171,678).

Another reported cationic lipid composition is a cationic cholesterolderivative (“DC-Chol”) which has been formulated into liposomes incombination with DOPE. (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). For certain cell lines, these liposomeswere said to exhibit lower toxicity and provide more efficienttransfection than the DOTMA-containing compositions.

Lipopolylysine, made by conjugating polylysine to DOPE has been reportedto be effective for transfection in the presence of serum. (Zhou, X. etal., Biochim. Biophys. Acta 1065:8, 1991).

However, of the cationic lipids which have been proposed for use indelivering macromolecules to cells, no particular cationic lipid hasbeen reported to work well with a wide variety of cell types. Since celltypes differ from one another in membrane composition, differentcationic lipid compositions and different types of lipid aggregates maybe effective for different cell types, either due to their ability tocontact and fuse with target cell membranes directly or due to differentinteractions with intracellular membranes or the intracellularenvironment. For these and other reasons, design of effective cationiclipids has largely been empirical. In addition, to content and transfer,other factors believed important include, for example, ability to formlipid aggregates suited to the intended purpose, toxicity of thecomposition to the target cell, stability as a carrier for themacromolecule to be delivered, and function in an in vivo environment.Thus, there remains a need for improved cationic lipids which arecapable of delivering macromolecules to a wide variety cell types withgreater effeciency.

SUMMARY OF THE INVENTION

In one aspect of the present invention novel amide-based cationic lipidshaving the structure:

or a salt, or solvate, or enantiomers thereof are provided wherein; (a)Y is a direct link or an alkylene of 1 to about 20 carbon atoms; (b) R₁is H or a lipophilic moiety; (c) R₂, R₃, and R₄ are positively chargedmoieties, or at least one but not all of R₂, R₃, or R₄ is a positivelycharged moiety and the remaining are independently selected from H, analkyl moiety of 1 to about 6 carbon atoms or a heterocyclic moiety; (d)n and p are independently selected integers from 0 to 8, such that thesum of n and o is from 1 to 16; (e) X⁻ is an anion or polyanion and (f)m is an integer from 0 to a number equivalent to the positive charge(s)present on the lipid; provided that if Y is a direct link and the sum ofn and p is 1 then one of either R₃ or R₄ must have an alkyl moiety of atleast 10 carbon atoms.

In one embodiment R₁ may be a variety of lipophilic moieties including astraight chain alkyl moiety of 1 to about 24 carbon atoms, a straightchain alkenyl moiety of 2 to about 24 carbon atoms, a symmetricalbranched alkyl or alkenyl moiety of about 10 to about 50 carbon atoms, aunsymmetrical branched alkyl or alkenyl moiety of about 10 to about 50carbon atoms, a steroidyl moiety, a amine derivative, a glycerylderivative, or OCH(R₅R₆) or N(R₅R₆), wherein R₅ and R₆ are straightchain or branched alkyl moieties of about 10 to about 30 carbon.

In another embodiment when R₂, R₃, or R₄ are positively charged moietiesit is preferable that the positively charged moiety be an alkylaminemoiety, a fluoroalkylamine moiety, or a perfluoroalkylamine moiety of 1to about 6 carbon atoms, an arylamine moiety or an aralkylamine moietyof 5 to about 10 carbon atoms, a guanidinium moiety, an enamine moiety,a cyclic amine moiety, an amidine moiety, an isothiourea moiety, and asubstituted heterocyclic amine moiety, a substituted heterocyclic moietyor a substituted alkyl moiety of 1 to about 6 carbon atoms substitutedwith a substituent selected from the group consisting of NH₂, C(═O)NH₂,NHR₇, C(═O)NHR₇, NHR₇R₈, or C(═O)NHR₇R₈, wherein R₇ and R₈ areindependently selected from an alkyl moiety of 1 to about 24 carbonatoms, an alkenyl moiety of 2 to about 24 carbon atoms, an aryl moietyof about 5 to about 20 carbon atoms, and an aralkyl moiety of about 6 toabout 25 carbon atoms.

When any of R₂, R₃, or R₄ are not a positively charged moiety it ispreferable that the at least one but not all of R₂, R₃, or R₄ areindependently selected from a substituted heterocyclic moiety of 1 toabout 6 carbon atoms, or a substituted alkyl moiety of 1 to about 6carbon atoms substituted with substituents selected from OH, thio, arylof 1 to about 20 carbon atoms, or OR₇, wherein R₇ is an alkyl moiety of1 to about 24 carbon atoms, an alkenyl of 2 to about 24 carbon atoms, anaryl of about 5 to about 20 carbon atoms or an aralkyl of about 6 toabout 25 carbon atoms.

It is particularly prefered that when R₂, R₃, or R₄ is an arylaminemoiety that it be tryptophane, phenylanaline, or tyrosine.

In another prefered embodiment R₂ is an amino acid residue having apositively charged side chain wherein the amino group(s) may beoptionally substituted with an alkyl of 1 to about 6 carbon atoms orsubstituted to form a secondary, tertiary, or quaternary amine with analkyl moiety of 1 to about 6 carbon atoms optionally substituted withsubstituents selected from hydroxyl, amino, alkoxy moiety of 1 to about6 carbon atoms, alkylamino moiety of 1 to about 6 carbon atoms, ordialkylamino moiety of 2 to about 12 carbon atoms.

Preferably when R₁ is steroidyl moiety it is a cholesteryl moiety.

It is preferable when R₂is an amino acid that it be lysine, arginine,histidine, ornithine, or an amino acid analog. In particular, when R₂ isan amino acid analog it is preferable that it be 3-carboxyspermidine,5-carboxyspermidine, 6-carboxyspermine, or monoalkyl, dialkyl, orperalkyl substituted derivatives which are substituted on one or moreamine nitrogens with an alkyl group of 1 to about 6 carbon atoms.

It is prefereable that R₃ and R₄ independently be a lipophilic moiety of1 to about 24 carbon atoms, a positively charged moiety, or a negativelycharged moiety. In particular, when both or either R₃ and R₄ are alipohilic moiety it is preferable that it be a straight chain alkylmoiety of 1 to about 24 carbon atoms, a straight chain alkenyl moiety of2 to about 24 carbon atoms, a symmetrical branched alkyl or alkenylmoiety of about 10 to about 50 carbon atoms, a unsymmetrical branchedalkyl or alkenyl moiety of about 10 to about 50 carbon atoms, an arylmoiety of about 5 to about 20 carbon atoms, an aralkyl moiety of about 6to about 25 carbon atoms, or a steroidyl moiety.

When both or either R₃ and R₄ are a positively charged moiety it ispreferable that the moiety be an amino acid residue having a positivelycharged group on the side chain, an alkylaminoalkyl moiety, afluoroalkylaminoalkyl moiety, a perfluoroalkylaminoalkyl moiety, aguanidiniumalkyl moiety, an enaminoalkyl moiety, a cyclic aminoalkylmoiety, an amidinoalkyl moiety, an isothiourea alkyl moiety, or aheterocyclic amine moiety.

It is also preferable that when both or either R₃and R₄ are a negativelycharged moiety it be a carboxyalkyl moiety, a phosphonoalkyl moiety, asulfonoalkyl moiety, or a phosphatidylalkyl moiety of 1 to about 24carbon atoms.

It is further prefered that the sum of the integers n and p be from 1 to8, more preferably from 1 to 4 and most preferably from 1 to 2.

It is also preferable that X⁻ be a pharmaceutically acceptable anion orpolyanion.

In a particularly prefered embodiment the amide-based cationic lipid hasthe structure

In another aspect of the present invention compositions comprising apolyanionic macromolecule and any of the lipids described above areprovided. In particular, the polyanionic macromolecule may be a varietyof molecules including an expression vector capable of expressing apolypeptide in a cell. In a prefered embodiment the polyanionicmacromolecule is an oligomucleotide or an oligomer and most preferablyDNA.

In still another aspect of the invention methods for the delivery of apolyanionic macromolecule into a cell by contacting any of thecompositions above with the cell are provided. In particular, a methodis provided to interfere with the expression of a protein in a cell bycontacting any of the the compositions described above with a cellwherein the composition comprises an oligomer having a base sequencethat is substantially complimentary to an RNA sequence in the cell thatencodes the protein.

The present invention further provides a kit for delivering apolyanionic macromolecule into a cell comprising any of the compositionsdescribed above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts synthetic schemes for the preparation of compounds 1-9and1-10. In this figure, i denotes hydroxylamine hydrochloride; ii denotesRaney nickel, H₂ at 45 psi, and 1:1 dichloromethane:methanol; iiidenotes benzyl chloroformate, aqueous sodium hydroxide, water andtoluene; iv denotes dicyclohexylcarbodiimide (“DCC”),1-hydroxybenzotriazole (“HOBT” or “HOBt”) and tetrahydrofuran (“THF”); vdenotes palladium on carbon, H₂ gas at 50 psi and 9:1dichloromethane:methanol; vi denotesN,N²-bis-[(1,1-dimethylethoxy)carbonyl]-L-ornithine,N-hydroxysuccinimidyl ester; vii denotes compound 1-6; and viii denotestrifluoroacetic acid and dichloromethane.

FIG. 2 depicts a synthetic scheme for the preparation of the compound2-5. In this figure, i denotes dioctadecylamine (“DODA”), DCC, and HOBTin dichloromethane; ii denotes trifluoroacetic acid and1,2-dichloroethane (“DCE”) to give a quantitative (99%) yield ofcompound 2-2; iii denotes compound 1-6,N²,N⁵-bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[3-[(1,1-dimethylethoxy)carbonyl]amino-propyl]-L-ornithine, DCCand HOBT to give a 77% yield of compound 2-3; iv denotes 10% palladiumon carbon and H₂ at 55 psi to give a 97% yield of compound 2-4; and vdenotes 7.0 M HCl in dioxane to give a quantitative yield of compound2-5.

FIG. 3 depicts a synthetic scheme for the preparation of compound 3-5.In this figure, i denotes DODA, DCC and HOBT in dichloromethane to givea 85% yield of compound 3-1; ii denotes deprotection using 1:1trifluoroacetic acid:dichloromethane to give a quantitative compound3-2; iii denotes compound 1-6, triethylamine (“TEA”) and DCM to give a60% yield of compound 3-3; iv denotes catalytic hydrogenation usingpalladium on carbon and H₂ at 55 psi to give an 85% yield of compound3-4; and v denotes Boc deprotection using 1:1 TFA:DCM to give aquantitative yield of compound 3-5.

FIG. 4 depicts a reaction scheme for the preparation of compound 4-5. Inthis figure, i denotes DODA, DCC and HOBT in DCM to give a quantitative(95%) yield of compound 4-1; ii denotes TFA to give a quantitative yieldof compound 4-2; iii denotes tetra-Boc-carboxy-spermidine, TEA and DCMto give a 27% yield of compound 4-3 (67% of unreacted lipid (compound4-2) was isolated); iv denotes palladium on carbon and H₂ at 50 psi togive a 53% yield of compound 4-4; and v denotes Boc deprotection withTFA to give a 94% yield of compound 4-5.

FIG. 5 depicts a reaction scheme for the preparation of compound 5-6. Inthis figure, i denotes DODA, DCC and HOBT in DCM to give an 85% yield ofcompound 2-1; ii denotes palladium on carbon with H₂ at 55 psi to givean 88% yield of compound 5-1; iii denotes L-glutamic acidbis(phenylamethyl)ester, toluene sulfonic acid salt, DCC, HOBT, TEA, andDCM to give a 85% yield of compound 5-2; iv denotes deprotection withTFA and DCE to give a quantitative yield of compound 5-3; v denotescompound 1-6 in DCM to give a 90% yield of compound 5-4; vi denotespalladium on carbon with H₂ at 55% psi to give 95% yield of compound5-5; and vii denotes Boc deprotection with TFA and DCE to give aquantitative yield of compound 5-6.

FIG. 6 depicts a reaction scheme for the preparation of compound 6-5. Inthis figure, i denotes DODA, DCC, and NHS in DCM to give n 70% yield ofcompound 6-2; ii denotes Pd/C in 2:1 methanol:DCM to give a 66% yield ofcompound 6-3; iii denotes TetraBoc carboxyspermine, DCC and HOBt in DCMto give a 20% yield of compound 6-4; and iv denotes TFA to give aquantitative yield of compound 6-5. In connection with compounds 6-1 and6-2 “Z” represents benzyloxycarbonyl.

FIG. 7 depicts the structure of a cationic lipid, compound 7-1. Thiscompound and its synthesis is described in the commonly assigned andco-pending U.S. patent application, “Novel Methylphosphonate-BasedCationic Lipids”, U.S. Ser. No. 08/484,716, filed Jun. 7, 1995, thedisclosure of which is incorporated herein by reference.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to first set forth definitions of certain termsthat will be used herein after. These terms will have the followingmeanings unless explicitly stated otherwise:

The term “lipophilic moiety” refers to a moiety which demonstrates oneor more of the following characteristics: tend to be water insoluble,tend to be soluble in non-polar solvent, tend to favor octanol inoctanol/water partition measurements, or tend to be compatible withlipid bilayers and may be bilayer forming.

The phrase “charged moiety” as used in the terms “positively chargedmoiety” and “negatively charged moiety” refers to a moiety, independentof the cationic lipid for which it is a substituent, having a netpositive or negative charge respectively within the pH range of 2 to 12.The net charge for the cationic lipid is the summation of all chargedmoieties occurring on the lipid, such that the net charge may bepositive, neutral or negative.

The term “alkyl” refers to saturated aliphatic groups includingstraight-chain, branched-chain and cyclic groups. Suitable alkyl groupsinclude, but are not limited to, cycloalkyl groups such as cyclohexyland cyclohexylmethyl. “Lower alkyl” refers to alkyl groups of 1 to 6carbon atoms. Fluoroalkyl or perfluoroalkyl refers to singly, partially,or fully fluorinated alkyl groups.

The term “alkenyl” refers to an unsaturated aliphatic group having atleast one double bond.

The term “arylamine” refers to aromatic groups that have at least onering having a conjugated pi electron system and includes carbocyclicaryl, heterocyclic aryl and biaryl groups, all of which may beoptionally substituted.

The term “aralkylamine” refers to an alkyl group substituted with anaryl group. Suitable aralkyl groups include benzyl, picolyl, and thelike, all of which may be optionally substituted.

The term “aralkyl” refers to an alkyl group substituted with an arylgroup. Suitable aralkyl groups include benzyl, picolyl, and the like,all of which may be optionally substituted.

The term “oligonucleoside” or “oligomer” refers to a chain ofnucleosides that are linked by internucleoside linkages that isgenerally from about 4 to about 100 nucleosides in length, but which maybe greater than about 100 nucleosides in length. They are usuallysynthesized from nucleoside monomers, but may also be obtained byenzymatic means. Thus, the term “oligomer” refers to a chain ofoligonucleosides that have internucleosidyl linkages linking thenucleoside monomers and, thus, includes oligonucleotides, nonionicoligonucleoside alkyl- and aryl-phosphonate analogs, alkyl- andaryl-phosphonothioates, phosphorothioate or phosphorodithioate analogsof oligonucleotides, phosphoramidate analogs of oligonucleotides,neutral phosphate ester oligonucleoside analogs, such asphosphotriesters and other oligonucleoside analogs and modifiedoligonucleosides, and also includes nucleoside/non-nucleoside polymers.The term also includes nucleoside/non-nucleoside polymers wherein one ormore of the phosphorus group linkages between monomeric units has beenreplaced by a non-phosphorous linkage such as a formacetal linkage, athioformacetal linkage, a morpholino linkage, a sulfamate linkage, asilyl linkage, a carbamate linkage, an amide linkage, a guanidinelinkage, a nitroxide linkage or a substituted hydrazine linkage. It alsoincludes nucleoside/non-nucleoside polymers wherein both the sugar andthe phosphorous moiety have been replaced or modified such as morpholinobase analogs, or polyamide base analogs. It also includesnucleoside/non-nucleoside polymers wherein the base, the sugar, and thephosphate backbone of the non-nucleoside are either replaced by anon-nucleoside moiety or wherein a non-nucleoside moiety is insertedinto the nucleoside/non-nucleoside polymer. Optionally, saidnon-nucleoside moiety may serve to link other small molecules which mayinteract with target sequences or alter uptake into target cells.

“Lipid aggregate” is a term that includes liposomes of all types bothunilamellar and multilamellar as well as micelles and more amorphousaggregates of cationic lipid or lipid mixed with amphipathic lipids suchas phospholipids. “Target cell” refers to any cell to which a desiredcompound is delivered, using a lipid aggregate as carrier for thedesired compound.

“Transfection” is used herein to mean the delivery of expressiblenucleic acid to a target cell, such that the target cell is renderedcapable of expressing said nucleic acid. It will be understood that theterm “nucleic acid” includes both DNA and RNA without regard tomolecular weight, and the term “expression” means any manifestation ofthe functional presence of the nucleic acid within the cell, includingwithout limitation, both transient expression and stable expression.

“Delivery” is used to denote a process by which a desired compound istransferred to a target cell such that the desired compound isultimately located inside the target cell or in, or on the target cellmembrane. In many uses of the compounds of the invention, the desiredcompound is not readily taken up by the target cell and delivery vialipid aggregates is a means for getting the desired compound into thecell. In certain uses, especially under in vivo conditions, delivery toa specific target cell type is preferable and can be facilitated bycompounds of the invention.

All references which have been cited below are hereby incorporated byreference in their entirety.

The generic structure of functionally active cationic lipids requiresthree contiguous moities, e.g. cationic-head-group, a linker, and alipid-tail group. While a wide range of structures can be envisioned foreach of the three moieties, it has been demonstrated that there is no apriori means to predict which cationic lipid will successfully transfectanionic macromolecules into a particular cell line. The property of acationic lipid to be formulated with an anionic macromolecule which willthen successfully transfect a cell line is empirical. We demonstrate theabilities of novel cationic lipids which are chemically linked intomultimeric constructions to enhance the uptake of macromolecules.

The novel amide-based cationic lipids of the present invention have thegeneral structure:

comprising any salt, solvate, or enantiomers thereof. The symbols R₁,R₂, R₃, R₄, Y, X, n, and m are described as follows; R₁ represents thelipid-tail group of the amide-based cationic lipid and may be hydrogenor a variety of lipophilic moieties, in particular, these include forexample, a straight chain alkyl of 1 to about 24 carbon atoms, astraight chain alkenyl of 2 to about 24 carbon atoms, a symmetricalbranched alkyl or alkenyl of about 10 to about 50 carbon atoms, aunsymmetrical branched alkyl or alkenyl of about 10 to about 50 carbonatoms, a amine derivative, a steroidyl moiety, a glyceryl derivative,and OCH(R₅R₆) or N(R₅R₆), wherein R₅ and R₆ are straight chain orbranched alkyl moieties of about 10 to about 30 carbon atoms.

When R₁ is an amine derivative a variety of such derivatives may beutilized, for example, a straight chain alkylamine moiety of 1 to about24 carbon atoms, a straight chain alkenylamine moiety of 2 to about 24carbon atoms, a symmetrical branched alkylamine or alkenylamine moietyof about 10 to about 50 carbon atoms, a unsymmetrical branchedalkylamine or alkenylamine moiety of about 10 to about 50 carbon atoms,cyclic amine moiety of about 3 to about 10 carbon atoms, or a steroidylmoiety.

In the case where R₁ is a steriodal moiety a variety of such moietiesmay be utilized including for example pregnenolone, progesterone,cortisol, corticosterone, aldosterone, androstenedione, testosterone, orcholesterol or analogs thereof.

R₂ represents the cationic head group of the amide-based cationic lipidand may be a positively charged moiety independent of R₃ and R₄. In suchcase, R₂ may be an amino acid residue having a unsubstituted orsubstituted positively charged side chain. Where the amino acid residueis substituted the substituent may be an alkyl of 1 to about 6 carbonatoms or a substituent which renders a secondary, tertiary, orquaternary amine having an alkyl moiety of 1 to about 6 carbon atomswhich is substituted with a hydroxyl, an amino, an alkoxy of 1 to about6 carbon atoms, an alkylamino of 1 to about 6 carbon atoms, or adialkylamino of 2 to about 12 carbon atoms.

In particular, when R₂ is an amino acid residue it may be, for example,lysine, arginine, histidine, ornithine, or an amino acid analog.Specific examples of amino acid analogs include 3-carboxyspermidine,5-carboxyspermidine, 6-carboxyspermine and a monoalkyl, dialkyl, orperalkyl substituted derivative which is substituted on one or moreamine nitrogens with an alkyl group of 1 to about 6 carbon atoms.

R₂, R₃, and R₄ may be positively charged moieties, or at least one butnot all of R₂, R₃, or R₄ may be a positively charged moiety. In thelatter case, the remaining R group(s) may independently be a hydrogen, asubstituted or unsubstituted alkyl moiety of 1 to about 6 carbon atoms,or a substituted or unsubstituted heterocyclic moiety of about 5 toabout 10 carbon atoms. More specifically, these groups may besubstituted with a hydroxyl, a thio, an aryl of 1 to about 20 carbonatoms, or OR₇, wherein R₇ is an alkyl moiety of 1 to about 24 carbonatoms, an alkenyl of 2 to about 24 carbon atoms, an aryl of about 5 toabout 20 carbon atoms or an aralkyl of about 6 to about 25 carbon atoms.

When R₂, R₃, and R₄ are positively charged moieties they mayindependently be, for example, an alkylamine moiety, a fluoroalkylaminemoiety, or a perfluoroalkylamine moiety of 1 to about 6 carbon atoms, anarylamine moiety or an aralkylamine moiety of 5 to about 10 carbonatoms, a guanidinium moiety, an enamine moiety, a cyclic amine moiety,an amidine moiety, an isothiourea moiety, a heterocyclic amine moiety,or a substituted heterocyclic moiety or a substituted alkyl moiety of 1to about 6 carbon atoms substituted with a substituent selected from thegroup consisting of NH₂, C(═O)NH₂, NHR₇, C(═O)NHR₇, NHR₇R₈, andC(═O)NHR₇R₈, wherein R₇ and R₈ are independently selected from an alkylmoiety of 1 to about 24 carbon atoms, an alkenyl moiety of 2 to about 24carbon atoms, an aryl moiety of about 5 to about 20 carbon atoms, and anaralkyl moiety of about 6 to about 25 carbon atoms. In particular, whenat least R₂, R₃, or R₄is an arylamine moiety a variety of such moietiesmay be utilized including, for example, tryptophane, phenylanaline, andtyrosine.

R₃ and R₄ may also be independently a lipophilic moiety or a negativelycharged moiety. In particular, when R₃ and/or R₄ is a lipophilic moietyit may be a straight chain alkyl moiety of about 3 to about 24 carbonatoms, a straight chain alkenyl moiety of 2 to about 24 carbon atoms, asymmetrical branched alkyl or alkenyl moiety of about 10 to about 50carbon atoms, a unsymmetrical branched alkyl or alkenyl moiety of about10 to about 50 carbon atoms, an aryl moiety of about 5 to about 20carbon atoms, an aralkyl moiety of about 6 to about 25 carbon atoms, ora steroidyl moiety.

In the case when R₃ and/or R₄ is a negatively charged moiety it may be acarboxyalkyl moiety, a phosphonoalkyl moiety, a sulfonoalkyl moiety, ora phosphatidylalkyl moiety of 1 to about 24 carbon atoms.

The linker comprises the structure joining the head group, R₁ to thelipid-tail group, R₂. This structure includes Y may be a direct linkfrom —(C═O)— to —(CHR₃)— or an alkylene of 1 to about 20 carbon atoms.

n and p are integers indicating the number of repeating units enclosedby the brackets and having magnitudes independent from each otherranging from 0 to 8, such that the sum of n and p is from 1 to 16, inparticular cases the sum of the integers range from 1 to 4 and inspecific instances from 1 to 2.

The counterion represented by X⁻ is an anion or a polyanion that bindsto the positively charged groups present on the phosphonic acid-basedcationic lipid via charge-charge interactions. When these cationiclipids are to be used in vivo the anion or polyanion should bepharmaceutically acceptable.

m is an integer indicating the number of anions or polyanions associatedwith the cationic lipid. In particular this integer ranges in magnitudefrom 0 to a number equivalent to the positive charge(s) present on thelipid.

In particular, when Y is a direct link and the sum of n and p is 1, thenone or either R₃ or R₄ must comprise an alkyl moiety of at least 10carbon atoms.

The cationic lipids of the present invention include salts, solvates, orenatiomeric isomers resulting from any or all asymmetric atoms presentin the lipid. Included in the scope of the invention are racemicmixtures, diastereomeric mixtures, optical isomers or synthetic opticalisomers which are isolated or substantially free of their enantiomericor diasteriomeric partners. The racemic mixtures may be separated intotheir individual, substantially optically pure isomers by techniquesknown in the art, such as, for example, the separation of diastereomericsalts formed with optically active acid or base adjuncts followed byconversion back to the optically active substances. In most instances,the desired optical isomer is synthesized by means of stereospecificreactions, beginning with the appropriate stereoisomer of the desiredstarting material. Methods and theories used to obtain enriched andresolved isomers have been described (Jacques et al., “Enantiomers,Racemates and Resolutions.” Kreiger, Malabar, Fla, 1991).

The salts include pharmaceutically or physiologically acceptablenon-toxic salts of these compounds. Such salts may include those derivedby combination of appropriate cations such as alkali and alkaline earthmetal ions or ammonium and quaternary amino ions with the acid anionmoiety of the phosphate or phosphorothioate acid group present inpolynucleotides. Suitable salts include for example, acid addition saltssuch as HCl HBr, HF, HI, H₂SO₄, and trifluoroacetate. The salts may beformed from acid addition of certain organic and inorganic acids, e.g.,HCl Hbr, H₂SO₄, amino acids or organic sulfonic acids, with basiccenters, (e.g. amines), or with acidic groups. The composition hereinalso comprise compounds of the invention in their un-ionized, as well aszwitterionic forms.

Exemplary invention cationic lipids have the structures shown in theSummary of the Invention above.

The cationic lipids form aggregates with polyanionic macromolecules suchas oligonucleotides, oligomers, peptides, or polypeptides throughattraction between the positively charged lipid and the negativelycharged polyanionic macromolecule. The aggregates may comprisemultiamellar or unilamellar liposomes or other particles. Hydrophobicinteractions between the cationic lipids and the hydrophobicsubstituents in the polyanionic macromolecule such as aromatic and alkylmoieties may also facilitate aggregate formation. Cationic lipids havebeen shown to efficiently deliver nucleic acids and peptides into cellsand are suitable for use in vivo or ex vivo.

Cationic lipid-polyanionic macromolecule aggregates may be formed by avariety of methods known in the art. Representative methods aredisclosed by Felgner et al., supra; Eppstein et al. supra; Behr et al.supra; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al.Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci.75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984;Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization is used whenconsistently small (50 to 200 nm) and relatively uniform aggregates aredesired (Mayhew, E., supra) . In general aggregates may be formed bypreparing lipid particles consisting of either (1) a cationic lipid ofthe invention or (2) a cationic lipid mixed with a colipid, followed byadding a polyanionic macromolecule to the lipid particles at about roomtemperature (about 18 to 26° C.). In general, conditions are chosen thatare not conducive to deprotection of protected groups. The mixture isthen allowed to form an aggregate over a period of about 10 minutes toabout 20 hours, with about 15 to 60 minutes most conveniently used. Thecomplexes may be formed over a longer period, but additional enhancementof transfection efficiency will not usually be gained by a longer periodof complexing. Colipids may be neutral or synthetic lipids having no netcharge or a positive or a negative charge. In particular, naturalcolipids that are suitable for preparing lipid aggregates with thecationic lipids of the present invention aredimyristoylphosphatidylethanolamine,dipalmitoyl-phosphatidylethanolamine,palmitoyloleolphosphatidyl-ethanolamine, cholesterol,distearoyalphosphatidyl-ethanolamine, phosphatidylethanolaminecovalently linked to polyethylene glycol and mixtures of these colipids.

The optimal cationic lipid:colipid ratios for a given cationic lipid isdetermined by mixing experiments to prepare lipid mixtures foraggregation with a polyanionic macromolecule using cationiclipid:colipid ratios between about 1:0.1 and 1:10. Methods to determineoptimal cationic lipid:colipid ratios have been described (see, Felgner,infra). Each lipid mixture is optionally tested using more than oneoligonucleotide-lipid mixture having different nucleic acid:lipid molarratios to optimize the oligonucleotide:lipid ratio.

Suitable molar ratios of cationic lipid:colipid are about 0.1:1 to1:0.1, 0.2:1 to 1:0.2, 0.4:1 to 1:0.4, or 0.6:1 to 1:0.6. Lipid particlepreparation containing increasing molar proportions of colipid have beenfound to enhance oligonucleotide transfection into cells with increasingcolipid concentrations.

In addition, the cationic lipids can be used together in admixture, ordifferent concentrations of two or more cationic lipids in admixture,with or without colipid.

Liposomes or aggregates maybe conveniently prepared by first drying thelipids in solvent (such as chloroform) under reduced pressure. Thelipids may then be hydrated and converted to liposomes or aggregates byadding water or low ionic strength buffer (usually less than about 200mM total ion concentration) followed by agitating (such as vortexingand/or sonication) and/or freeze/thaw treatments. The size of theaggregates or liposomes formed range from about 40 nm to 600 nm indiameter.

The amount of an oligonucleotide delivered to a representative cell byat least some of the cationic lipids was found to be significantlygreater than the amount delivered by commercially available transfectionlipids. The amount of oligonucleotide delivered into cells was estimatedto be about 2- to 100-fold greater for the cationic lipids of theinvention based on the observed fluorescence intensity of transfectedcells after transfection using a fluorescently labeled oligonucleotide.The cationic lipids described herein also transfect some cell types thatare not detectably transfected by commercial lipids. Functionality ofcationic lipid-DNA aggregates was demonstrated by assaying for the geneproduct of the exogenous DNA. Similarly, the functionality of cationiclipid-oligonucleotide aggregates were demonstrated by antisenseinhibition of a gene product.

The cationic lipids described herein also differed from commerciallyavailable lipids by efficiently delivering an oligonucleotide into cellsin tissue culture over a range of cell confluency from about 50 to 100%.Most commercially available lipids require cells that are at arelatively narrow confluency range for optimal transfection efficiency.For example, Lipofectin™ requires cells that are 70-80% confluent fortransfecting the highest proportion of cells in a population. Thecationic lipids described herein may be used to transfect cells that areabout 10-50% confluent, but toxicity of the lipids was more pronounced,relative to that seen using cells that are about 50-100% confluent. Ingeneral, the cationic lipids transfected cells that were about 60-100%confluent with minimal toxicity and optimal efficiency. Confluencyranges of 60-95% or 60-90% are thus convenient for transfectionprotocols with most cell lines in tissue culture.

The cationic lipid aggregates were used to transfect cells in tissueculture and the RNA and the DNA encoded gene products were expressed inthe transfected cells.

The cationic lipid aggregates may be formed with a variety ofmacromolecules such as oligonucleotides and oligomers. Oligonucleotidesused in aggregate formation may be single stranded or double strandedDNA or RNA, oligonucleotide analogs, and plasmids.

In general, relatively large oligonucleotides such as plasmids or mRNAswill carry one or more genes that are to be expressed in a transfectedcell, while comparatively small oligonucleotides will comprise (1) abase sequence that is complementary (via Watson Crick or Hoogsteenbinding) to a DNA or RNA sequence present in the cell or (2) a basesequence that permits oligonucleotide binding to a molecule inside acell such as a peptide, protein, or glycoprotein. Exemplary RNAs includeribozymes and antisense RNA sequences that are complementary to a targetRNA sequence in a cell.

An oligonucleotide may be a single stranded unmodified DNA or RNAcomprising (a) the purine or pyrimidine bases guanine, adenine,cytosine, thymine and/or uracil: (b) ribose or deoxyribose; and (c) aphosphodiester group that linkage adjacent nucleoside moieties.Oligonucleotides typically comprise 2 to about 100 linked nucleosides.Typical oligonucleotides range in size from 2-10, 2-15, 2-20, 2-25,2-30, 2-50, 8-20, 8-30 or 2-100 linked nucleotides. Oligonucleotides areusually linear with uniform polarity and, when regions of invertedpolarity are present, such regions comprise no more than one polarityinversion per 10 nucleotides. One inversion per 20 nucleotides istypical. Oligonucleotides can also be circular, branched ordouble-stranded. Antisense oligonucleotides generally will comprise asequence of about from 8-30 bases or about 8-50 bases that issubstantially complementary to a DNA or RNA base sequence present in thecell. The size of oligonucleotide that is delivered into a cell islimited only by the size of polyanionic macromolecules that canreasonably be prepared and thus DNA or RNA that is 0.1 to 1 Kilobase(Kb), 1 to 20 Kb, 20 Kb to 40 Kb or 40 Kb to 1,000 Kb in length may bedelivered into cells.

Oligonucleotides also include DNA or RNA comprising one or more covalentmodifications. Covalent modifications include (a) substitution of anoxygen atom in the phosphodiester linkage of an polynucleotide with asulfur atom, a methyl group or the like, (b) replacement of thephosphodiester group with a nonphosphorus moiety such as —O—CH₂O—,—S—CH₂O— or —O—CH₂O—S, and (c) replacement of the phosphodiester groupwith a phosphate analog such as —O—P(S) (O)—O, —O—P(S) (S)—O—, —O—P(CH₃) (O)—O or —O—P (NHR¹⁰) (O) —O— where R¹⁰ is alkyl of 1 to about 6carbon atoms, or an alkyl ether of 1 to about 6 carbon atoms. Suchsubstitutions may constitute from about 10% to 100% or about 20% toabout 80% of the phosphodiester groups in unmodified DNA or RNA. Othermodifications include substitutions of or on sugar moiety such asmorpholino, arabinose 2′-fluororibose, 2′-fluoroarabinose,2′-O-methylribose, or 2′-O-allylribose. Oligonucleotides and methods tosynthesize them have been described (for example see PCT/US90/03138,PCT/US90/06128, PCT/US90/06090, PCT/US90/06110, PCT/US92/03385,PCT/US91/08811, PCT/US91/03680, PCT/US91/06855, PCT/US91/01141,PCT/US92/10115, PCT/US92/10793, PCT/US93/05110, PCT/US93/05202,PCT/US92/04294, WO 86/05518, WO 89/12060, WO 91/08213, WO 90/15065, WO91/15500, WO 92/02258, WO 92/20702, WO 92/20822, WO 92/20823, U.S. Pat.No.: 5,214,136 and Uhlmann Chem Rev. 90:543, 1990).

The linkage between the nucleotides of the oligonucleotide may be avariety of moieties including both phosphorus-containing moieties andnon phosphorus-containing moieties such as formacetal, thioformacetal,riboacetal and the like. A linkage usually comprises 2 or 3 atomsbetween the 5′ position of a nucleotide and the 2′ or 3′ position of anadjacent nucleotide. However, other synthetic linkers may containgreater than 3 atoms.

The bases contained in the oligonucleotide may be unmodified or modifiedor natural or unnatural purine or pyrimidine bases and may be in the αor β anomer form. Such bases may be selected to enhance the affinity ofoligonucleotide binding to its complementary sequence relative to basesfound in native DNA or RNA. However, it is preferable that modifiedbases are not incorporated into an oligonucleotide to an extent that itis unable to bind to complementary sequences to produce a detectablystable duplex or triplex.

Exemplary bases include adenine, cytosine, guanine, hypoxanthine,inosine, thymine, uracil, xanthine, 2-aminopurine, 2,6-diaminopurine,5-(4-methylthiazol-2-yl)uracil, 5-(5-methylthiazol-2-yl)uracil,5-(4-methylthiazol-2-yl)cytosine, 5-(5-methylthiazol-2-yl)cytosine andthe like. Other exemplary bases include alkylated or alkynylated baseshaving substitutions at, for example, the 5 position of pyrimidines thatresults in a pyrimidine base other than uracil, thymine or cytosine,(i.e., 5-methylcytosine, 5-(1-propynyl)cytosine, 5-(1-butynyl)cytosine,5-(1-butynyl)uracil, 5-(1-propynyl)uracil and the like). The use ofmodified bases or base analogs in oligonucleotides have been previouslydescribed (see PCT/US92/10115; PCT/US91/08811; PCT/US92/09195; WO92/09705; WO 92/02258; Nikiforov, et al., Tet. Lett. 33:2379, 1992;Clivio, et al., Tet. Lett. 33:65, 1992; Nikiforov, et al., Tet. Lett.32:2505, 1991; Xu, et al., Tet. Lett. 32:2817, 1991; Clivio, et al.,Tet. Lett. 33:69, 1992; and Connolly, et al., Nucl. Acids Res. 17:4957,1989).

Aggregates may comprise oligonucleotides or oligomers encoding atherapeutic or diagnostic polypeptide. Examples of such polypeptidesinclude histocompatibility antigens, cell adhesion molecules, cytokines,antibodies, antibody fragments, cell receptor subunits, cell receptors,intracellular enzymes and extracellular enzymes or a fragment of any ofthese. The oligonucleotides also may optionally comprise expressioncontrol sequences and generally will comprise a transcriptional unitcomprising a transcriptional promoter, an enhancer, a transcriptionalterminator, an operator or other expression control sequences.

Oligonucleotides used to form aggregates for transfecting a cell may bepresent as more than one expression vector. Thus, 1, 2, 3, or moredifferent expression vectors may be delivered into a cell as desired.Expression vectors will typically express 1, 2, or 3 genes whentransfected into a cell, although many genes may be present such as whena herpes virus vector or a artificial yeast chromosome is delivered intoa cell. Expression vectors may further encode selectable markers (e.g.neomycin phosphotransferase, thymidine kinase, xanthine-guaninephosphoribosyl-transferase, and the like) or biologically activeproteins such as metabolic enzymes or functional proteins (e.g.immunoglobulin genes, cell receptor genes, cytokines (e.g. IL-2, IL-4,GM-CSF, γ-INF and the like), or genes that encode enzymes that mediatepurine or pyrimidine metabolism and the like)).

The nucleic acid sequence of the oligonulcleotide coding for specificgenes of interest may be retrieved, without undue experimentation, fromthe GenBank of EMBL DNA libraries. Such sequences may include codingsequences, for example, the coding sequences for structural proteins,hormones, receptors and the like, and the DNA sequences for other DNAsof interest, for example, transcriptional and translational regulatoryelements (promoters, enhancers, terminators, signal sequences and thelike), vectors (integrating or autonomous) and the like. Non-limitingexamples of DNA sequences which may be introduced into cells includethose sequences coding for fibroblast growth factor (see WO 87/01728);ciliary neurotrophic factor (Lin et al., Science, 246:1023, 1989); humaninterferon-α receptor (Uze, et al., Cell, 60:225, 1990); theinterleukins and their receptors (reviewed in Mizal, FASEB J., 3:2379,1989); hybrid interferons (see EPO 051,873); the RNA genome of humanrhinovirus (Callahan, Proc. Natl. Acad. Sci., 82:732, 1985); antibodiesincluding chimeric antibodies (see U.S. Pat. No.: 4,816,567); reversetranscriptase (see Moelling, et al., J. Virol., 32:370, 1979); human CD4and soluble forms thereof (Maddon et al., Cell, 47:333, 1986), WO88/01304 and WO 89/01940); and EPO 330,191, which discloses a rapidimmunoselection cloning method useful for the cloning or a large numberof desired proteins.

Aggregates can be used in antisense inhibition of gene expression in acell by delivering an antisense oligonucleotide into the cell (seeWagner, Science 260:1510, 1993 and WO 93/10820). Such oligonucleotideswill generally comprise a base sequence that is complementary to atarget RNA sequence that is expressed by the cell. However, theoligonucleotide may regulate intracellular gene expression by binding toan intracellular nucleic acid binding protein (see Clusel, Nucl. AcidsRes. 21:3405, 1993) or by binding to an intracellular protein ororganelle that is not known to bind to nucleic acids (see WO 92/14843).A cell that is blocked for expression of a specific gene(s) is usefulfor manufacturing and therapeutic applications. Exemplary manufacturinguses include inhibiting protease synthesis in a cell to increaseproduction of a protein for a therapeutic or diagnostic application(e.g., reduce target protein degradation caused by the protease).Exemplary therapeutic applications include inhibiting synthesis of cellsurface antigens to reduce rejection and/or to induce immunologictolerance of the cell either after it is implanted into a subject orwhen the cell is trnsfected in vivo (e.g. histocompatibility antigens,such as MHC class II genes, and the like).

Methods to introduce aggregates into cells in vitro and in vivo havebeen previously described (see U.S. Pat. Nos.: 5,283,185 and 5,171,678;WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol Chem 269:2550,1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human GeneTher. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J.11:417, 1992.

Entry of liposomes or aggregates into cells may be by endocytosis or byfusion of the liposome or aggregate with the cell membane. When fusiontakes place, the liposomal membrane is integrated into the cell membraneand the aqueous contents of the liposome merge with the fluid in thecell.

Endocytosis of liposomes occurs in a limited class of cells; those thatare phagocytic, or able to ingest foreign particles. When phagocyticcells take up liposomes or aggregates, the cells move the spheres intosubcellular organelles known as lysosomes, where the liposomalmembranbes are thought to be degraded. From the lysosome, the liposomallipid components probably migrate outward to become part of cell'smembranes and other liposomal components that resist lysosomaldegradation (such as modified oligonucleotides or oligomers) may enterthe cytoplasm.

Lipid fusion involves the transfer of individual lipid molecules fromthe liposome or aggregate into the plasma membrane (and vice versa); theaqueous contents of the liposome may then enter the cell. For lipidexchange to take place, the liposomal lipid must have a particularchemistry in relation to the target cell. Once a liposomal lipid joinsthe cell membrane it can either remain in the membrane for a period oftime or be redistributed to a variety of intracellular membranes. Thecationic lipids of the present invention can be used to deliver anexpression vector into a cell for manufacturing or therapeutic use. Theexpression vectors can be used in gene therapy protocols to deliver atherapeutically useful protein to a cell or for delivering nucleic acidsencoding molecules that encode therapeutically useful proteins orproteins that can generate an immune response in a host for vaccine orother immunomodulatory purposes according to known methods (see U.S.Pat. Nos.: 5,399,346 and 5,336,615, WO 94/21807 and WO 94/12629). Thevector-transformed cell can be used to produce commercially useful celllines, such as a cell line for producing therapeutic proteins or enzymes(e.g. erythropoietin, and the like), growth factors (e.g. human growthhormone, and the like) or other proteins. The aggregates may be utilizedto develop cell lines for gene therapy applications in humans or otherspecies including murine, feline, bovine, equine, ovine or non-humanprimate species. The aggregates may be to deliver polyanionicmacromolecules into cells in tissue culture medium in vitro or in ananimal in vivo.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES General Methods

All reactions were run under a positive pressure of dry argon. Reactionsrequiring anhydrous conditions were performed in flame-dried glasswarewhich was cooled under argon. Tetrahydrofuran (THF, Aldrich Milwaukee,Wis.) was distilled from potassium/benzophenone ketyl immediately priorto use. Methylene chloride, pyridine, toluene, heptane, methanol, andethanol were obtained as anhydrous reagent (<0.005% water) or reagentgrade and were used without further purification. TLC was performed on0.2 mm E. Merck precoated silica gel 60 F₂₅₄ TLC plates (20×20 cmaluminum sheets, Fisher, Pittsburgh, Pa.). Flash chromatography wasperformed using E. Merck 230-400 mesh silica gel. All ¹H, ¹³C and ³¹PNMR spectra were recorded on a 300 MHz Bruker ARX Spectrometer (Bruker,Boston, Mass.) and were obtained in CDCl₃ unless otherwise indicated.Mass spectra were provided by The Scripps Research Institute MassSpectrometry Facility of La Jolla, Calif. FAB mass spectra were obtainedon a FISONS VG ZAB-VSE double focusing mass spectrometer equipped with acesium ion gun (Fisions, Altrincham, UK). ESI mass spectra were obtainedon an API III PE Sciex triple-quadrupole mass spectrometer (Sciex,Toronto, Calif.).

Example 1 Synthesis ofL-Ornithylglycyl-N-(1-heptadecyloctadecyl)glycinamide,Dihydrotrifluoroacetate (1-9) andN²,N⁵-Bis(3-aminopropyl)-L-ornithylglycyl-N-(1-heptadecyloctadecyl)glycinamide,Tetrahydrotrifluoroacetate(1-10)

Synthesis of Stearone oxime (1-1) was prepared as follows and usedwithout further purification. A 500 mL, round-bottomed reaction flaskequipped with a magnetic stirrer, reflux condenser, and an argon inlettube was charged with stearone (12.3 g, (24.3 mmol)), hydroxylaminehydrochloride (8.5 g, (121.6 mmol)), pyridine (12.0 mL, (148.4 mmol)),and ethanol (125 mL). After the mixture was refluxed for 2 hours it wascooled to room temperature and left standing overnight. The resultingwhite solid was collected, washed with water and then with ethanol,air-dried for 30 minutes, and then dried under vacuum (0.5 mm Hg) atroom temperature for 15 hours to afford 11.59 g (91% yield) of compound1-1 as a white solid: mp 68-69° C. (lit.(Grun et al., Angew. Chem. 39,421, 1926) mp 66-67° C.); R_(f)0.49 (9:1 heptane:ethyl acetate); ¹HNMR_(—)δ7.40 (br s, 1H), 2.32 (t, J=7.8 Hz, 2H), 2.15 (t, J=7.8 Hz, 2H),1.49 (m, 4H), 1.25(br s, 56H), 0.88 (t, J=6.5 Hz, 6H).

Synthesis of 18-Pentatriacontanamine (1-2) was prepared as follows andused without further purification. A Parr bottle was charged withstearone oxime (5.0 g, (9.6 mmol)), 50 mL of a 1:1 mixture of methylenechloride:methanol, and 1 g of wet Raney nickel (washed with 95% ethanolto remove water). The reaction bottle was placed in a Parr shakerapparatus, filled with H₂ gas and evacuated by vacuum to remove air, andthen subjected to 45 psi H₂ gas at room temperature with shaking for 15hours. The reaction mixture was then filtered and washed with 1:1methylene chloride:methanol. The filtrate was concentrated by rotaryevaporation and the crude product was purified by flash chromatographyon silica gel (100 g) using 95:5 methylene chloride:methanol to afford1.99 g (41% yield) of 1-2 as a white solid and 1.22 g of a mixture(ratio not determined) of 1-2 and stearone. Coumpound 1-2 gave thefollowing spectral data: R_(f)0.58 (4:1 methylene chloride:methanol); ¹HNMR δ_(—)7.50-6.70 (br s, 2H), 3.03 (apparent quintet, J=6.3 Hz, 1H),1.62-1.05 (m, 64H), 0.88 (t, J=6.6 Hz, 6H); MS (ESI) m/z 509 (MH⁺).

Synthesis N-Carbobenzyloxyglycylglycine (1-3) was prepared as followsand used without further purification. A 250 mL round-bottomed reactionflask equipped with a magnetic stirrer was charged with glycylglycine(1.0 g, (7.6 mmol)), aqueous sodium hydroxide (8.3 mL of a 2 M solution,(16.6 mmol)), water (2.0 mL) and toluene (5.0 mL). The resultingbiphasic mixture was cooled in an ice-water bath and then benzylchloroformate (1.2 mL, (8.3 mmol)) was added to the reaction mixturedropwise via syringe. The reaction mixture was allowed to warm to roomtemperature and stirred for 3 hours. The reaction mixture was thentransferred to a separatory funnel and extracted with ethyl acetate. Thephases were separated and the aqueous phase was acidified to pH 2-3 bydropwise addition of a 6 N aqueous HCl solution. The acidified aqueousphase was placed in a refrigerator for 30 minutes. The resulting whitesolid was collected, air-dried and then vacuum-dried to afford 1.21 (60%yield) of compound 1-3 as a white solid. R_(f)0.44 (1:1 methylenechloride:methanol); ¹H NMR (CD₃OD) δ7.39-7.27 (m, 5H), 5.10 (s, 2H),3.97 (s, 2H), 3.82 (s, 2H).

Synthesis of (Carbobenzyloxy)glycyl-N-(1heptadecyl-octadecyl)glycinamide(1-4) was prepared as follows and used without further purification. A100 mL round bottomed reaction flask equipped with a magnetic stirrerwas charged with N-carbobenzyloxyglycylglycine (0.21 g, (0.78 mmol)),18-pentatriacontanamine (1-2) (0.40 g, (0.78 mmol)),dicyclo-hexylcarbodiimide (0.18 g, (0.86 mmol)), 1-hydoxybenzotriazolehydrate (0.12 g, (0.86 mmol)) and THF (10 mL). The reaction mixture wasstirred at room temperature under argon for 15 hours. The reactionmixture was filtered and washed with ethyl acetate. The filtrate wasconcentrated by rotary evaporation and the crude product was purified byflash chromatography on silica gel using 1:1 ethyl acetate-heptane toafford 0.39 g (66% yield) of compound 1-4 as a white solid. R_(f)0.22(1:1 ethyl acetate-heptane); ¹H NMR δ7.15 (t, J=4.5 Hz, 1H), 6.18 (d,J=9.6 Hz, 1H), 5.74 (t, J=4.5 Hz, 1H), 5.10 (s, 2H), 3.90 (m, 5H),2.20-1.00 (m, 64H), 0.88 (t, J=6.6 Hz, 6H); MS (FAB) m/z 757 (MH⁺).

Synthesis of Glycyl-N-(1-heptadecylocta decyl)-glycinamide (1-5) wasprepared as follows and used without further purification. A Parr bottlewas charged withN-(carbobenzyloxy)glycyl-N-(1-heptadecyloctadecyl)glycinamide (1-4)(2.95 g, (3.9 mmol)), palladium on carbon (0.5 g, 5% Pd content), and 50mL of a 9:1 mixture of methylene chloride:methanol. The reaction bottlewas secured in a Parr shaker apparatus, filled with H₂ gas, evacuated byvacuum twice to remove oxygen, and then subjected to 50 psi H₂ gas, withshaking, at room temperature for 10 hours. The reaction mixture wasfiltered through a filter agent and washed with 1:1 methylenechloride:methanol to remove catalyst. The filtrate was concentrated byrotary evaporation to afford 1.75 g (72% yield) of compound 1-5 as awhite solid. R_(f)0.42 (4:1 methylene chloride-methanol); ¹H NMR δ7.88(br s, 1H), 5.85 (br d, J=10.8 Hz, 1H), 4.02-3.79 (m, 3H), 3.40 (br s, 2H), 1.69 (br s, 3H), 1.58-0.98 (m, 63H), 0.88 (t, J=6.5 Hz, 6H).

Synthesis ofN²,N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[3-[1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine, N-hydroxysuccinimydyl ester (1-6) wasprepared as follows and used without further purification. A 100 mLround-bottomed reaction flask was charged withN²,N₅-Bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine(Behr, Acc. Chem. Res. 26:274, 1993; (2.08 g, (3.2 mmol)),dicyclohexylcarbodiimide (0.73 g, (3.5 mmol)), N-hydroxysuccinimide(0.41 g, (3.5 mmol)), and methylene:chloride (20 mL). The reactionmixture was stirred for 5 hours and then placed in a refrigerator (0-5°C.) overnight. The mixture was filtered and washed with methylenechloride. The filtrate was concentrated by rotary vaporation and thecrude product was purified by flash chromatography on silica gel using1:1 ethyl acetate-heptane to provide 1.2 g (50% yield) of compound 1-6as a white solid. ¹H NMR δ5.26 (br s, 1H), 4.77 (br s, 1H), 4.28 (br s,1H), 3.22-3.09 (m, 10H), 2.84 (s, 4H), 2.05-1.61 (m, 8H), 1.48 and 1.46and 1.44 (3 s, 36H); MS (ESI) m/z 744 (MH⁺).

Synthesis ofN²,N⁵-Bis-[(1,1-dimethyethoxy)carbonyl]-L-ornithylglycyl-N-(1-heptadecyloctadecyl)glycinamide(1-7) was prepared as follows and used without further purification. A50-mL round-bottomed reaction flask was charged with glycylglycinamide1-5 (0.05 g, (0.08 mmol)),N,N²-bis-[(1,1-dimethylethoxy)carbonyl]-L-ornithine,N-hydroxysuccinimidyl ester (0.04 g, (0.09 mmol)), and methylenechloride (3 mL). The reaction was stirred at room temperature for 1hour. The reaction mixture was concentrated by rotary evaporation andthen purified by flash chromatography on silica gel (10 g) using ethylacetate to afford 0.06 g (80% yield) of compound 1-7 as a white waxysemi-solid. R_(f)0.52 (9:1 methylene chloride:methanol); ¹H NMR δ7.66(br s, 1H), 7.43 (br s, 1H), 6.43 (br s, 1H), 5.55 (br s, 1H), 4.87 (brs, 1H), 4.21 (br s, 1H), 4.07-3.77 (m, 4H), 3.31-3.02 (m, 2H), 2.35 (brs, 1H), 1.93-1.00 (m, 68H) 1.43 (s, 18H), 0.88 (t, J=6.6 Hz, 6H).

Synthesis ofN²,N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithylglycyl-N-(1-heptadecyloctadecyl)glycinamide(1-8) was prepared as follows and used without further purification. A50-mL round-bottomed reaction flask was charged with glycylglycinamide1-5 (0.05 g, (0.08 mmol)),N²,N⁵-bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[-3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine,N-hydroxysuccinimydyl ester (1-6) (0.07 g, (0.09 mmol)), and methylenechloride (3 mL). The reaction was stirred at room temperature for 1hour. The reaction mixture was concentrated by rotary evaporation andthen purified by flash chromatography on silica gel (10 g) using ethylacetate to afford 0.055 g (55% yield) of compound 1-8 as a colorlessthick oil: R_(f) 0.57 (9:1 methylene chloride:methanol); ¹H NMRδ7.87-7.57 (m, 2H), 6.48 (m, 1H), 5.06 (br s, 1H), 4.45-3.38 (m, 6H),3.37-2.92 (m, 10H), 2.38-0.98 (m, 72H), 1.45 (s, 36H), 0.88 (t, J=6.6Hz, 6H); MS (ESI) m/z 1251 (MH⁺).

Compound 1-9 was synthesized according to the following procedure. To a50-mL round-bottomed reaction flask containing glycylglycinamide 1-7(0.07 g, (0.08 mmol)) was added methylene chloride (2.0 mL) and to thissuspension was added trifluoroacetic acid (0.5 mL). The reaction mixturebecame a homogeneous yellow solution and was left standing for 20minutes. The reaction mixture was concentrated by rotary evaporation andthen coevaporated with heptane (3×10 mL). The resulting residue wassubjected to high vacuum (0.1 mm Hg) at room temperature for 15 hours toafford 0.07 g (97% yield) of compound 1-9 as a pale yellow waxy solid:¹H NMR (CD₃OD) δ7.72 (d, J=9.0 Hz, 1H), 4.13 and 4.07 and 3.94 and 3.88(4 s, amide rotamers, 4H), 3.95-3.82 (m, 2H), 2.97 (m, 2H), 1.95-1.76(m, 4H), 1.48-1.27 (m, 64H), 0.89 (t, J=6.6 Hz, 6H); MS (ESI) m/z 694(MH⁺).

Compound 1-10 was synthesized according to the following procedure. To a50-mL round-bottomed reaction flask containing glycylglycinamide 1-8(0.06 g, (0.04 mmol)) was added methylene chloride (2.0 mL) and to thissuspension was added trifluoroacetic acid (0.5 mL). The reaction mixturebecame a homogeneous yellow solution and was left standing for 20minutes. The reaction mixture was concentrated by rotary evaporation andthen coevaporated with heptane (3×10 mL). The resulting residue wassubjected to high vacuum (0.1 mm Hg) at room temperature for 15 hours toafford 0.06 g (99% yield) of compound 1-10 as a pale yellow waxy solid:¹H NMR (CD₃OD) d 7.72 (d, J=9.0 Hz, 1H), 4.03 (s, 2H), 3.88 (s, 2H),4.03-3.79 (m, 2H), 3.16-3.02 (m, 10H), 2.20-1.05 (m, 72H), 0.89 (t,J=6.6 Hz, 6H) ; MS (ESI) m/z 851 (MH⁺).

Example 2

SynthesisN²-[N²,N⁵-Bis(3-aminopropyl)-L-ornithyl]-N,N-dioctadecyl-L-glutamine,tetrahydrochloride (2-5) Synthesis ofN²-[(1,1-Dimethylethoxy)carbonyl]-N,N-dioctadecyl-L-glutamine,phenylmethyl ester (2-1) was prepared as follows and used withoutfurther purification. To a solution of 5.0 g (14.8 mmol) ofBoc-Glu-a-OBn in 75 mL of dry DCM was added 8.5 g (16.3 mmol, 1.1equivalents) of dioctadecylamine (DODA), 3.4 g (4.6 mmol, 1.1equivalents) of DCC and 2.0 g of HOBT (14.8 mmol, 1.0 equivalents). Thereaction mixture was allowed to stir under argon atmosphere overnight.The precipitated dicyclohexylurea was filtered, the residue was washedwith 15 mL of DCM, and the combined filtrate was concentrated underreduced pressure to give a colorless oil. The crude product was purifiedby chromatography on silica gel (9:1 heptanes/ethyl acetate) to afford12.5 g (quantitative yield) of compound 2-1 as a colorless oil. Rf 0.28(4:1 heptanes:ethyl acetate) ¹HNMR δ: 7.35-7.30 (m, 5H), 5.44-5.42(broad d, J=6 Hz, 1H), 5.16 (AB q, J=12.3 Hz, 2H), 4.31-4.29 (m, 1H),3.31-3.21 (m, 2H), 3.13-3.08 (dd, J=8.1, 7.6 Hz, 2H) 2.38-2.01 (m, 4H),1.50-1.45 (m overlaps s, 13H) 1.26 (br s, 60H), 0.88 (t, J=6 Hz, 3H).

Synthesis of N,N-Dioctadecyl-L-glutamine, phenylmethyl ester,hydrotrifluoroacetate (2-2) was prepared as follows and used withoutfurther purification. To a solution of 12.5 (14.8 mmol)N²-[(1,1-dimethylethoxy)carbonyl]-N,N-dioctadecyl-L-glutamine,phenylmethyl ester 2-1 in 70 mL of 1,2-dichloroethane was added 50 mL ofTFA. The reaction mixture was allowed to stir at room temperature for 30minutes and concentrated under reduced pressure. The crude oil obtainedwas further coevaporated with heptane (4×50 mL) and left to stand underhigh vacuum overnight to afford 12.7 g (quantitative) of compound 2-2 asa waxy solid. ¹H NMR (CD,OD) δ: 7.23-7.14 (m, 5H), 5.07 (AB q, J=12.0Hz, 2H), 3.94 (t, J=6.6 Hz, 1H), 3.11-2.92 (m, 4H), 2.33 (t, J=7.5Hz,2H), 1.97 (dd, J=6.6, 12.0 Hz, 2H), 1.35-1.31 (m, 4H), 1.08 (m, 60H),0.69 (t, J=6.6 Hz, 3H).

Synthesis ofN²-[N²,N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[3-[((1,1-dimethylethoxy)-carbonyl)aminopropyl]-L-ornithyl]-N,N-dioctadyl-L-glutamine,phenylmethyl ether (2-3) was prepared as follows and used withoutfurther purification. To a solution of N,N-Dioctadecyl-L-glutamine,phenylmethyl ester, hydrotrifluoroacetate, compound 2-2, (3.75 g, 4.4mmol) in 40 mL of dry DCM, was added triethylamine (10 mL). The reactionmixture was allowed to stir at room temperature for 5 minutes andN²,N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[3-(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine(1-6A) (3.1 g, 4.8 mmol, 1.1 equivalents) was added in one portionfollowed by DCC (992 mg 4.8 mmol, 1.1 equivalents) and HOBt (300 mg, 2.2mmol, 0.5 equivalents). The progress of the reaction was followed by TLC(7:3 heptane:ethyl acetate), and was considered was complete after 3hours. The reaction mixture was concentrated under reduced pressure togive a crude oil that was purified by chromatography on silica gel (7:3heptane:ethyl acetate) to afford 4.6 g (77% yield) of compound 2-3 as acolorless oil. ¹H NMR δ: 7.33-7.29 (m, 5H), 5.25 9m, 1H), 5.14 (AB q,J=12.2 Hz), 4.47 (m, 1H), 3.23-3.06 (m, 14H), 1.44-1.31 (br , 40H),1.29-1.24 (br, 60H), 0.87 (t, J=6.6 Hz, 3H). Mass spec(ESI+) calcd for :1369, found 1370 (MH+).

Synthesis ofN²-[N²,N⁵-Bis[((1,1-dimethylethoxy)-carbonyl]-N²,N⁵-bis[3-[(1,1-dimethylethoxy)carbonyl]-aminopropyl]-L-ornithyl]-N-N-dioctadecyl-L-glutamine(2-4) was prepared as follows and used without further purification. Asolution of N²-[N²,N⁵-bis(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[3-[(1,1-dimethyl-ethoxy)carbonyl)aminopropyl]-L-ornithyl]-N,N-dioctadecyl-L-glutamine,phenylmethyl ether, compound 2-3, (4.5 g, 3.2 mmol) in 75 mL of ethylacetate was hydrogenated in presence 10% Pd/C (450 mg) under 55 psihydrogen in a Parr hydrogenator. After 12 hours the reaction mixture wasfiltered to remove the catalyst and concentrated to give compound 2-4 asa colorless oil (4.1 g, 97% yield). ¹H NMR δ: 9.49 (m, 1H), 4. 29 (m,3H), 3.31-3.23 (m, 16H), 1.28 (m, 60H), 0.89 (t, J=6.6 Hz, 6H).

Compound 2-5 was synthesized according to the following procedure. Tothe acid, compound 2-4 (3.7 g), was added warm dioxane (20 mL). Thissolution was cooled to room temperature and then was added 7.0 Msolution of HCl in dioxane (20 mL). The reaction mixture was thenallowed to stir at room temperature for 5 hours. It was thenconcentrated at reduced pressure to give a white solid which wascoevaporated with heptane (4×10 mL) to give compound 2-5 a white solid(quantitative yield). ¹H NMR (CD₃OD) δ: 4.50 (t, J=6.0 Hz, 1H), 4.15 (m,1H), 3.38-3.11 (m, 14H), 2.59-2.55 (m, 2H), 1,61-1.56 (m, 4H), 1.54 (br,60H), 0.90 (t J=6.0 Hz, 6H). Mass spec (ESI+) calcd for 879, found 880(MH+) (ESI−) found : 878.

Example 3 Synthesis of N²-[N²,N⁵-Bis(aminopropyl)-L-ornithyl]-N-N-dioctadecyl-L-a-glutamine,tetrahydrotrifluoroacetate (3-5)

Synthesis of4-[(1,1-dimethylethoxy)carbonyl]amino-5-(dioctadecyl)amino-5-oxopentanoicacid, phenylmethyl-ester (3-1) was prepared as follows and used withoutfurther purification. To a solution (338 mg, 1.0 mmol) ofBoc-Glu-g-benzyl ester in DCM (10 mL) was added dioctadecylamine (574 mg1.1 mmol), DCC (227 mg, 1.1 mmol) and HOBt (14 mg, 0.1 mmol). Thereaction mixture was allowed to stir at room temperature for 3 days andthen filtered. The filtrate was concentrated under reduced pressure togive a colorless oil that was purified by silica gel chromatography (4:1heptanes:ethyl acetate) to afford 717 mg (85% yield) of compound 3-1. ¹HNMR δ: 7.36-7.29 (m, 5H), 5.39-5.36 (m 1H), 5.13 (AB q, J=12.0 H), 4.66,4.61 (m,1H), 3.52-3.45 (m, 2H), 3.09-3.07 (m, 2H), 1.41 (s, 9H), 1.41(br, 60H), 0.88 (t, J=6.0 Hz, 6H).

Synthesis of 5-(Dioctadecyl)amino-5-oxopentanoic acid, phenylmethylester, hydrotrifluoroacetate (3-2) was prepared using standard Bocdeprotecting procedures (see, e.g., Example 2 using TFA:DCM (1:1)(quantitative yield). ¹H NMR δ: 7.56-7.36 (m, 5H), 5.18, (AB q, J=12.2Hz), 4.35 (br dd, J=4.2, 3.3 Hz. 1H), 3.57 (dd overlapping dd, 2H),3.22-3.12 (dd over laps dd, 2H), 2.60 (dd over laps dd 2H), 2.07-2.059m, 2H), 1.59-1.57(m, 4H), 1.27 9(br s, 60H), 0.88 (t, J=6.3 Hz, 6H).

Synthesis ofN²-[N²,N⁵-bis(1,1-dimethylethoxy)-carbonyl]-N²,N⁵-bis[3-[(1,1-dimethylethoxy)carbonyl]-aminopropyl]-L-ornithyl]-N,N-dioctadecyl-L-a-glutamine,phenylmethyl ester (3-3) was prepared as follows and used withoutfurther purification. (4S)-5-(Dioctadecyl)amino-5-oxopentanoic acid,phenylmethyl ester, hydrotrifluoroacetate (3-2) (285 mg 0.33 mmol) wascoupled with compound 1-6 (261 mg, 0.35 mmol) using protocol such asdescribed in Example 1. The crude product was purified by silica gelchromatography gave 200 mg (60% yield) of compound 3-3. (Also recoveredwas 150 mg of starting amine). ¹H NMR δ: 7.34-7.29 9m, 5H), 5.10 (br2H), 3.16-3.10 (m, 14H), 1.47 (m overlaps singlet, 40H), 1.24 (m, 60H),0.87 (t, J=6.3 Hz, 6H).

Synthesis ofN²-[N²,N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[3-[(1,1-dimethylethoxy)carbonyl]-aminopropyl]-L-ornithyl]-N-N-dioctadecyl-L-a-glutamine(3-4) was prepared by catalytic hydrogenation of 3-3 in EtOAc under 55psi using 10% Pd/C (85% yield) (see Example 2).

Compound 3-5 was synthesized according to the following procedure.Compound 3-4 was subjected to standard Boc deprotection using TFA:DCM(1:1) to give compound 3-5 in quantitative yield (see Example 1). ¹H NMR(CD₃OD/CDCl₃) δ: 4.01-3.98 (m, 1H), 3.59-3.57 (m 2H), 3.16-3.01 (m,12H), 3.01 (m, 2H), 1. 21(br m, 60H), 0.89 (t, J=6.6 HZ). Mass spec:calcd 879. found 880 (MH+).

Example 4 Synthesis ofN²-[N²,N⁵-Bis(3-aminopropyl)-L-ornithyl]-N,N-dioctadecyl-L-a-asparagine,tetrahydrotrifluoroacetate (4-5)

Synthesis of 3-Amino-4-(dioctadecyl)amino-4-oxobutanoic acid,phenylmethyl ester (4-1) was prepared as follows and used withoutfurther purification. Boc-Asp-b-benzyl ester was coupled withdioctadecylamine using DCC coupling procedures such as those describedin Examples 2. (quantitative yield of compound 4-1). ¹H NMR δ: 7.39-7.27(m, 5H), 5.28 (br d, J=12 Hz, 1H), 5.10, (AB q, J=12.0 Hz, 2H), 4.94,(dd, J=5.4, 6.0 Hz, 1H), 3.40-3.15 (m, 4H), 2.82 (dd, J=6.0, 15.6 Hz,1H), 2.63 (dd, J=5.9, 15.6 Hz, 1H), 1.47 (m overlaps s, 13 H), 1.25 (brm, 60H, 0.87 (t, J=6.3 Hz, 6H).

Synthesis of 3-Amino-4-(dioctadecyl)amino-4-oxobutanoic acid,phenylmethyl ester, hydrotrifluoroacetate (4-2) was prepared as followsand used without further purification.(3S)-3-Amino-4-(dioctadecyl)amino-4-oxobutanoic acid, phenylmethylester, compound 4-1, was exposed to TFA to remove Boc protecting group.A quantitative yield of compound 4-2 was obtained. ¹H NMR δ: 7.39 (m,5H), 5.19 (AB q, J=12.3 Hz, 2H), 4.55 (t, J=6.3, Hz, 1H), 3.54-3.10 (m,4H), 2.90 (d, J=6.1 Hz, 2H), 1.60-1.50 (m, 4H), 1.38 (m, 60H), 0.89 (t,J=6.6 Hz, 6H).

Synthesis ofN²-[N²,N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²-N⁵-bis[(1,1-dimethylethoxy)-carbonyl]aminopropyl]-L-ornithyl]-N,N-dioctadecyl-L-a-asparagine,phenylmethyl ester. (4-3) was prepared as follows and used withoutfurther purification. 3-Amino-4-(dioctadecyl)amino-4-oxobutanoic acid,phenylmethyl ester, hydrotrifluoroacetate (4-2) (420 mg, 0.5 mmol) wasdissolved in dry DCM (5 mL). To this solution was added TEA (1.0 mL);the reaction mixture was allowed to stir for 5 minutes, at which timetetra-Boc-carboxy spermine succinimidyl ester (369 mg, 0.5 mmol) wasadded in one portion. After 12 hours the reaction mixture wasconcentrated under reduced pressure and the crude product was purifiedby silica gel chromatography (7:3 heptanes: EtOAc) to afford 186 mg ofproduct (27% yield).

Synthesis of N²-[N²,N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²-N⁵-bis[3-[(1,1-dimethyl-ethoxy)carbonyl]aminopropyl]-L-ornithyl]-N-N-dioctadecyl-L-a-asparagine (4-4)was prepared as follows and used without further purification.N²-[N²,N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²-N⁵-bis[(1,1-dimethylethoxy)-carbonyl]aminopropyl]-L-ornithyl]-N,N-dioctadecyl-L-a-asparagine,phenylmethyl ester (4-3) (186 mg, 0.13 mmol) was hydrogenated usingstandard procedures (see Example 2) to afford 92 mg of product (53%yield).

Compound 4-5 was synthesized according to the following procedure.N²-[N²,N⁵-Bis[(1,1dimethylethoxy)carbonyl]-N²-N⁵-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl]-N-N-dioctadecyl-L-a-asparagine(4-4) was subjected to standard Boc deprotection conditions (see Example1). to give the product as a white waxy solid (94% yield) ¹H NMR δ: 3.84(t, J+4.5 Hz, 1H), 3.49-3.44 (m, 1H), 3.33-3.26 (m. 2H), 3.07-2.93 (m,12H), 2.68-2.59 (m, 2H), 2.04-1.68 (m, 12H), 1.19 (br m, 60H), 0.80 9tJ=5.4 Hz). Mass spec calcd: 865 found: 866 (MH+)

Example 5 Synthesis N-[N²-[N²,N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dioctadecyl-L-glutaminyl]-L-glutamicacid (5-6)

Synthesis of N²-(1,1-Dimethylethoxy)carbonyl-N-N-dioctadecyl-L-glutamine(5-1) was prepared as follows and used without further purification.N²-[1,1-Dimethylethoxy)carbonyl]-N,N-dioctadecyl-L-glutamine,phenylmethyl ester (2-1) (2.4 g, 2.85 mmol) was dissolved in 30 mL ofethyl acetate and 500 mg of 10% Pd/C was added to this solution. Theresulting mixture was hydrogenated in a Parr hydrogenation apparatus at55 psi for 12 hours. The catalyst was filtered and the filtrate wasconcentrated to give the product, compound 5-1, as colorless oil.

Synthesis ofN-[N²-(1,1-Dimethylethoxy)carbonyl-N-N-dioctadecyl-L-glutaminyl]-L-glutamicacid, bis(phenylmethyl)ester (5-2) was prepared as follows and usedwithout further purification.N²-(1,1-Dimethylethoxy)carbonyl-N-N-dioctadecyl-L-glutamine 5-1, (680mg, 0.9 mmol) was coupled with L-glutamic acid, bis (phenylmethyl)ester,toluene sulfonic acid salt (450 mg, 0.9 mmol) using standard DCC, HOBtmediated coupling (see Examples 2 and 3) to afford 812 mg of product(85% yield). ¹H NMR δ: 7.88 (m, 1H), 7.38-7.32 (m, 10H), 5.83 (br, 1H),5.17, 5.12 (2 apparent s, 4H), 4.68 (m, 1H), 4.15 (dd, J=4.6, 6 Hz, 1H),3.30-3.18 (m, 4H), 2.48-2.42 (m, 5H), 2.1-1.09, (m, 4H), 1.55-1.53 (m,4H), 1.20 (br, 60H), 0.91 (t, J=6.6 Hz).

Synthesis of N-(N,N-Dioctadecyl-L-glutaminyl]-L-glutamicacid,bis(phenylmethyl) ester, hydrotrifluoro-acetate (5-3) was preparedas follows and used without further purification.N-[N²-(1,1-Dimethylethoxy)carbonyl-N-N-dioctadecyl-L-glutaminyl]-L-glutamicacid, bis(phenylmethyl)ester (5-2) was subjected to standard TFAdeprotection (see, e.g., Example 2). This product was used in Example 5with out further characterization.

Synthesis ofN-[N²-[N²,N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²-N⁵-bis[3-[1,1-dimethylethoxy)-carbonyl]aminopropyl]-L-ornithyl]-N-N-dioctadecyl]-L-glutaminyl]-L-glutamicacid,bis(phenylmethyl)ester (compound 5-4) was prepared as follows andused without further purification. Compound 5-3 (200 mg, 0.19 mmol) wascoupled with N² N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine,N-hydroxysuccinimydyl ester (1-6) (156 mg, 0.21 mmol) in DCM (5 mL).After 6 hours the reaction mixture was concentrated under reducedpressure and the crude product thus obtained was purified by silica gelchromatography (10% MeOH in DCM) to afford 270 mg of product as a whitefoamy solid. (90% yield) ¹H NMR δ: 8.71 (m, 1H), 8.32 9m, 1H), 7.35-7.29(m, 10H), 5.35 (m, 2H), 5.20-5.11 (m, 4H), 4.39-4.37 (q, J=6.1 Hz),3.36-3.10 (m, 14H), 1.45 (br s, 36H), 1.27 (br, 60H), 0.89 (t, J=6.5 Hz,6H).

Synthesis of N-[N²-[N²,N⁵-Bis(3-aminopropyl)-L-ornithyl]-N,N-dioctadecyl-L-glutaminyl]-L-glutamicacid, Tetrahydrotrifluoroacetate (5-5) was prepared as follows and usedwithout further purification. Compound 5-4 (200 mg, 0.12 mmol) washydrogenated with catalytic Pd/C at 55 psi (95% yield). This compoundwas carried to next stage with out further characterization and/orisolation.

Compound 5-6 was synthesized according to the following procedure.Compound 5-5 (100 mg) was subjected to standard Boc deprotection usingTFA (see Example 1). Obtained was the product as dirty white waxy solid(quantitative yield). 1H NMR (CDCl₃/CD₃OD) δ: 7.36 (d, J=3.0 Hz, 1H),4.54 (dd, J=4.7, 9.0 Hz, 1H), 4.41 (t, J=7 Hz, 1H), 4.00 (m, 1H),3.37-3.30 (m, 14H), 1.57 (m, 4H), 1.29 (br m, 60H), 0.88 (t, J=6.6 HZ,6H). Mass spec(ESI±): calcd : 1008, found 1009 (MH+) and 1007 (M−H+)

Example 6 Synthesis of Compound 6-5

Synthesis of compound 6-2 was prepared as follows and used withoutfurther purification. To a solution of 6-benzyloxycarbonyl amino capriocacid, (6-1), (300 mg, 1.1 mmol) in 10 mL of DCM, was addeddioctadecylamine (DODA) (590 mg, 1.1 mmol), DCC (233 mg, 1.1 mmol) andN-hydroxysuccinimide (“NHS”) (143 mg, 1.1 mmol). The reaction mixturewas warmed to enable the starting material to completely dissolve. Itwas then allowed to stir under argon atmosphere for 2 days; then theprecipitated DC urea was filtered off. The filtrate was washed with 1NHCl, and then saturated NaHCO₃ solution, dried over MgSO₄, filtered andconcentrated to afford 620 mg (71% yield) of a viscous oil. ¹H NMR δ:7.36-7.30 (m, 5H), 5.43 (m, 2H), 4.84 (br m 1H), 3.49-319, (m, 6H), 2.25(t, J=7.5 Hz, 1H), 1.67-1.50 (m, 6H), 1.26 (br s, 60H), 0.88 (t, J=6.0Hz, 6H).

Synthesis of Compound 6-3 was prepared as follows and used withoutfurther purification. Compound 6-2 (620 mg, 0.62 mmol) was dissolved in15 mL of a 2:1 mixture of methanol:DCM and 100 mg 10% Pd/C was added.This mixture was subjected to hydrogenation at 35 psi for 12 hours. Thereaction mixture was then filtered to remove the catalyst andconcentrated to afford 420 mg of Compound 6-3 as a waxy solid. ¹H NMR δ:3.25-3.01 (overlapping m, 6H), 2.28 (t, J=7.2 Hz, 2H), 1.81 (m, 2H),1.64-1.59 (m, 10H), 1.23 (br s, 60H), 0.83 (t, J=6.6 Hz, 6H).

Synthesis of Compound 6-4 was prepared as follows and used withoutfurther purification. To a solution of amine, Compound 6-3, (195 mg, 0.3mmol) in 5 mL of DCM, was added tetraboc carboxyspermine (220 mg, 0.34mmol) and HOBt (50 mg, 0.37 mmol). To this solution was then added asolution of DDC (70 mg, 0.34 mmol) in 3 mL of DCM. The reaction mixturewas allowed to stir overnight. After usual DCC coupling workup, a crudemixture was obtained that was then purified by chromatography on silicagel (13 g) with hexane:ethyl acetate (4:6) as eluent to afford 75 mg ofproduct, Compound 6-4, as a glassy solid. 1H NMR δ: 4.38 (br, 1H),3.24-3.11 (m, 16H), 2.26 (t, J=7.2 H, 2H), 1.57 (overlapping s and m,50H), 1.24 (br s, 60H), 0.86 (t J=6.6 Hz, 6H). Mass spectometrycalculated 1263, observed 1264 (mH+), 1266 (m+Na⁺).

Compound 6-5 was synthesized according to the following procedure.Compound 6-4 (4 mg) was subjected to standard Boc deprotectingconditions and workup conditions using trifluoroacetic acid (seeExample 1) to give a quantitative yield of the product compound 6-5 as apale yellow waxy solid. ¹H NMR δ: 3.3-29 (m, 12H), 2.4-2.2 (m, 20H),1.25 (br s, 60H), 0.85 (t, 6H).

Example 7 Preparation and Transfection Protocols for COS-7, SNB-19, RDand C8161 Cells with Mixtures of Cationic Lipids and CAT Plasmid

A. Culturing and Transfection of Cells

Cell lines were plated at 1.5×10⁵ cells/well in a 12 well plate formaton the day before tranfection. Cultures were maintained at 37° C. in 5%CO₂. On the next day, when the cells reached approximately 80%confluence, the transfection mixes were prepared as follows: 126 μg ofthe target CAT plasmid pG1035 (described below) was added to 36.0 mL ofOpti-MEM® (Gibco/BRL, Gaithersburg, Md.) to make a plasmid stocksolution. 63 μg of each lipid mix (from a high concentration stock in100% ethanol) was added to individual 1.5 mL aliquots Opti-MEM® andmixed thoroughly. Then, 2 mL of the DNA stock (containing 7 μg ofplasmid) were added to each 1.5 mL aliquot of lipid/Opti-MEM® and gentlyvortexed. This procedure yielded 3.5 mL of plasmid/lipid mixture at 2μg/mL plasmid and 18 μg/mL lipid for a 9 to 1 lipid to DNA ratio. Thequantity of ethanol in the final cell cultures was 2% or less. Thissmall quantity of ethanol was confirmed to have no adverse effect on anyof the cell lines.

In order to prepare cells for transfection, the culture medium was thenaspirated out of the wells and the cells were rinsed twice in 1 mLOpti-MEM® per well. The transfection experiments were performed intriplicate; thus, 1 mL of each transfection mix was then added to eachof three wells. The cells were cultured in the transfection mix for 5 to6 hours. The transfection mix was then removed and replaced with 1 mL ofcomplete culture medium (DMEM or DMEM/F12 plus 10% fetal bovine serumand 1:100 dilution of penicillin:streptomycin stock, all from Gibco/BRL,(Gaithersburg, Md.) and the cells were allowed to recover overnightbefore expression the CAT gene was measured.

Cell lysates were prepared by rinsing twice in PBS and then were treatedwith 0.5 mL of 1× Reporter Lysis Buffer (Promega, Madison, Wis.). Thelysed cells were pipetted into 1.5 mL tubes and frozen in CO₂/EtOH onceand thawed. The crude lysate was then clarified by microcentrifugationat 14,000 rpm for 10 minutes to pellet cell debris. The clearsupernatant was recovered and assayed directly or stored at −20° C. forassay later.

The cell lysates were then assayed for CAT activity and the totalprotein concentration was determined as described below in Example 7.The CAT activity was normalized to total protein and plotted as shown.

B. Chloramphenicol Acetyltransferase Assay

This assay was performed generally as follows. First, the followingreaction mixture was prepared for each sample:

65 mL 0.23 M Tris, pH 8/0.5% BSA (Sigma, St. Louis, Mo.), 4 mL¹⁴C-chloramphenicol, 50 nCi/mL (Dupont, Boston, Mass.), and 5 mL mg/mLn-butyryl coenzyme A (Pharmacia, Piscataway, N.J.).

A CAT activity standard curve was prepared by serially diluting CATstock (Promega, Madison, Wis.) 1:1000, 1:10,000 and 1:90,000 in 0.25 MTris, pH 8:0.5% BSA. The original stock CAT was at 7000 Units/mL. CATlysate was then added in a labeled tube with Tris/BSA buffer for finalvolume of 50 mL.

Approximately 74 mL of reaction mixture was then added to each sampletube, which was then typically incubated for approximately 1 hour in a37° C. oven. The reaction was terminated by adding 500 mL pristane:mixedxylenes (2:1) (Sigma, St. Louis, Mo.) to each tube. The tubes were thenvortexed for 2 minutes and spun for 5 minutes. Approximately 400 mL ofthe upper phase was transferred to a scintillation vial with 5 mLScintiverse (Fisher, Pittsburgh, Pa.). The sample was then counted in ascintillation counter (Packard).

C. Preparation of Plasmid pG1035

The plasmid pG1035 was used for transient transfections of COS-7 (ATCC #CRL-1651), SNB-19, C8161 and RD (ATCC # CCL-136) cells. pG1035 consistsof a modified CAT (choramphenicol acetyl transferase) gene (namedSplicerCAT) inserted into the eukaryotic vector pRc/CMV (Invitrogen, SanDiego, Calif.). SplicerCAT was created by inserting an artificial introninto the wild type CAT gene sequence in a plasmid named pG1036 (alsobased on pRc/CMV) using the polymerase chain reaction. Transfectionresults are set forth in Tables IA to ID.

(i) Description of inserted CAT sequences of plasmids pG1035 and pG1036.The sequences of the synthetic splice sites of pG1035 (SplicerCAT) andpG1036 (wild-type CAT) are set forth below:

(a) Partial sequence of the wild type CAT gene used to create plasmidpG1036:

[SEQ. ID. NO.1]                      +409 +410                        | | GCC UAU UUC CCU AUU UCC CUA AAG GGU UUA UUGAGA AUA

(b) Full sequence of the intron inserted within the CAT coding sequenceof pG1036 to create the SplicerCAT gene and plasmid pG1035:

[SEQ. ID. NO.2]                      +409 1                         | |... ACC UGG CCU AUU UCC CUA AAG{circumflex over ( )}gug agu gac uaa cuaagu                           39                            | cga cugcag acu agu cau ug(a) uug agu gua aca aga ccg                         87+410                           | | gau auc uuc gaa ccu cuc ucu cuc ucucag{circumflex over ( )}GGU UUA UUG AGA...

(ii) Preparation of Plasmids

The region of the CAT gene into which the intron was inserted is shownas the mRNA in sequence (a) above. The wild type CAT gene (Pharmacia)was inserted into pRc/CMV (Invitrogen, San Diego, Calif.) via Hind IIIrestriction sites to create plasmid pG1036. Bases +409 and +410 arelabeled on SEQ. ID. NOS. 1 and 2 for comparison of sequences only topG1035. A synthetic intron, shown in sequence (b) above, was insertedinto the CAT DNA to create plasmid pG1035. Mature mRNA sequences areshown uppercase, intronic sequences are lower case. The canonicalguanosine of the splice donor is labeled +409, which corresponds to base+409 of the CAT open reading frame. The first base of the intron islabeled 1. The canonical branchpoint adenosine is base 39 and thecanonical intronic splice acceptor guanosine is base 87 of the intron.Base +410 marks the resumption of the CAT open reading frame. Thesequences against which the oligomers are targeted are underlined. Theconsensus splice site bases are given in bold face italics (Smith etal., Ann. Rev. Genet. 23: 527, 1989; and Green, Ann. Rev. Genet. 20:671, 1986).

The clone pG1035 was assembled from two fragments created usingphosphorylated synthetic DNA PCR primers and plasmid pG1036 as the PCRtemplate in two separate PCR reactions. One reaction produced a HindIII-Spe I 5′-fragment containing the first ⅔ of the open reading frameand half of the synthetic intron. The second PCR reaction produced anSpe I-Not I fragment containing the second half of the intron and thelast ⅓ of the open reading frame. These PCR products were combined withHind III-Not I cut pRc/CMV in a 3-way ligation to yield the finalplasmid. The artificial CAT gene containing the intron is namedSplicerCAT. References applicable to the foregoing include Smith et al.,supra and Green supra.

D. Coomassie Protein Assay

The total protein content of the clarified cell lysates was determinedby mixing 6 μL of each cell lysate to 300 mL of Coomassie protein assayreagent (Pierce, Rockford, Md.) in the wells of an untreated microtiterassay plate. Concentration curve standards were prepared using 6 μL of0, 75, 100, 200, 250, 400, 500, 1000, and 1500 mg/mL BSA stock solutionsand 300 mL of the Coomassie reagent. The assay samples were allowed tosit for approximately 30 minutes before reading the optical absorbanceat 570 nm in a microplate reader (Molecular Probes).

The cells were assayed for CAT protein as described above. The resultsof the transfection efficiency of the cationic lipids are tabulated inTables IA to ID.

Example 8 FITC-Oligonucleotide Uptake Assay

A. Oligomers Used

The oligonucleotides used for the determination of cationic lipidmediated oligonucleotide uptake in all cell lines tested were:

[SEQ. ID NO. 1] #3498-PS: 5′ FITC-ggt-ata-tcc-agt-gat-ctt-ctt-ctc,

Oligomer 3498-PS has an all-phosphorothioate backbone. Thisoligonucleotide has 23 negative charges on the backbone and isconsidered to be 100% negatively charged.

[SEQ. ID NO. 2] #3498: 5′ FITC-ggt-ata-tcc-agt-gat-ctt-ctt-ctc,

Oligomer 3498 is a chimeric oligonucleoside. The underlined bases werelinked by a phosphorothioate backbone, while the other linkages in theoligomer consisted of alternating methylphosphonates andphosphodiesters. The oligomer had 11 methylphonate, 7 diester, and 5phosphorothioates linkages. The total charge density was 57% of 3498-PS.

[SEQ. ID NO.3] #3793-2: 5′ FITC-ggu-aua-ucc-agu-gau-cuu-cut,

Oligomer 3293-2 has an alternating methylphosphonate and diesterbackbone with all 2′-O-methyl groups on each ribose in theoligonucleotide. The total charge density was 50% of 3498-PS.

Stocks of oligomers 3498-PS and 3498 were prepared at 300 micromolar,while the oligomer 3793-2 stock was prepared at 440 micromolar.

B. Reagents and Cells

The commercially available lipids used in the assays were:

Lipofectin® (“LFN”) Lot#EF3101 1 mg/mL, Gibco/BRL (Gaithersburg, Md.)

LipofectAMINE® (“LFA”) Lot#EFN101 2 mg/mL, Gibco/BRL (Gaithersburg, Md.)

Transfectam® (“TFM”) Lot#437121 1 mg dry, Promega, (Madison, Wis.) andresuspended in 100% ethanol.

The novel lipids of the present invention used in these evaluations, aslisted in the data tables (Tables II A to C), were at 1 mg/mL in 100%ethanol.

The tissue culture cell stocks, SNB-19 (human glioblastoma), C8161 (ahuman amelanotic melanoma), RD (human rhabdomyosarcoma, ATCC # CCL-136)and COS-7 (African green monkey kidney cells, ATCC # CRL-1651) weremaintained in standard cell culture media: DMEM:F12 (1:1) mix fromMediatech, Lot#150901126, 10% fetal bovine serum from GeminiBioproducts, Lot#A1089K, 100 units/mL penicillin and 100 micrograms/mLstreptomycin, from Mediatech, Lot#30001044 and 365 micrograms/mLL-glutamine. The cells were maintained under standard conditions (37°C., 5% CO₂ atmosphere) at all times prior to fixation and microscopicexamination.

C. Preparation of Cells and Transfection Mixes

For each FITC labeled oligomer delivery determination, the appropriatecells were plated into 16 well slides (Nunc #178599, glass microscopeslide with 16 removable plastic wells attached to the slide surface witha silicone gasket) according to standard tissue culture methods. Eachcell line was plated at a starting density (approximately 20,000cells/well) that allowed them to be healthy and 60-80% confluent one totwo days after plating. The cells to were allowed to adhere to the glassand recover from the plating procedure in normal growth medium for 24 to48 hours before beginning the transfection procedure.

Oligonucleotide transfection mixes were made up in Opti-MEM® withoutantibiotics as follows: 500 μL aliquots of Opti-MEM® containing a 0.25micromolar solution of either oligomer 3498-PS, 3498, or 3793-2 (2micrograms of oligomer per sample) were pipetted into 1.5 mL Eppendorftubes. Cationic lipid or lipid mixture was then added to the oligomersolution to give a final 9:1 or 6:1 ratio (18 or 12 μg of lipid total)of cationic lipid to oligomer by weight, as listed in Tables IIA to IIC.The tubes were mixed by vortexing immediately after the addition oflipid.

Prior to beginning the transfection reactions the cells were rinsed in200 μL Opti-MEM®; then, the cells were rinsed with Dulbecco's phosphatebuffered saline (PBS) solution, and 200 μL of oligomer transfection mixwas added directly to a well to begin each transfection reaction.Transfection reactions were allowed to continue for four to six hours.

At that time, the cells were then rinsed in PBS from Mediatech and fixedfor ten minutes in 200 μL of 3.7% formaldehyde (Sigma, St. Louis, Mo.)to terminate the transfection reaction. Then the wells were rinsed againin PBS. The formaldehyde was quenched with 200 μL of 50 mM glycine(Sigma, St. Louis, Mo.) for ten minutes. Finally, the wells were thenemptied by shaking out the glycine solution.

At that time, the plastic chambers and silicone gasket were removed andthe cells were covered with Fluoromount-G mounting medium (from Fisher,Pittsburgh, Pa., with photobleaching inhibitors) and a cover slip.

Intracellular fluorescence was evaluated under 200× magnification with aNikon Labophot-2 microscope with an episcopic-fluorescence attachment.Using this equipment we could distinguish extracellular from nuclear andendosomal fluorescence.

The cells were scored for uptake of FITC labelled oligomer as follows:No nuclear fluorescence, 0; up to 20% fluorescent nuclei, 1; up to 40%fluorescent nuclei, 2; up to 60% fluorescent nuclei, 3; up to 80%fluorescent nuclei, 4; and up to 100% fluorescent nuclei, 5.

The results of the transfections in COS-7, SB-19, C-8161 and RD cellsare tabulated in Tables IIA to IIC.

TABLE 1 Transient transfection efficiency of cationic lipids in COS-7,SNB-19, RD and C8161 cells Lipid Mean SDV REL A. Cell line COS-7 None894 23 0 Transfectam 89437 14746 0.52 Lipofectamine 119902 7350 0.7Lipofectin 57084 4288 0.33 2-5 55598 4643 0.33 2-5/DOPE 159419 185640.93 2-5/7-1 162560 9944 0.95 1-9/DOPE 170801 5457 1 1-10 148040 39320.87 1-10/DOPE 138397 11512 0.81 B. Cell line SNB-19 None 807 24 0Transfectam 106060 17596 0.56 Lipofectamine 143064 12699 0.76 Lipofectin177312 3487 0.94 2-5 103280 12908 0.55 2-5/DOPE 134725 13224 0.712-5/7-1 172245 99236 0.91 1-9/DOPE 187651 5480 1 1-10 166179 18702 0.881-10/DOPE 188468 2650 1 C. Cell line RD None 743 32 0 Transfectam 512551490 0.29 Lipofectamine 85689 9618 0.48 Lipofectin 128481 8972 0.72 2-573921 3839 0.41 2-5/DOPE 104283 6701 0.58 2-5/7-1 178331 4630 1 1-9/DOPE123060 5312 0.69 1-10 124232 5248 0.7 1-10/DOPE 42824 2629 0.24 D. Cellline C8161 None 851 32 0 Transfectam 141138 2049 0.71 Lipofectamine133571 5823 0.67 Lipofectin 144780 11981 0.73 2-5 137710 16610 0.692-5/DOPE 199253 5307 1 2-5/7-1 153079 13322 0.77 1-9/DOPE 61088 80870.31 1-10 159578 6067 0.8 1-10/DOPE 84229 7287 0.42

TABLE II Demonstration of nuclear delivery of oligonucleotides of varingcharge densities by novel cationic lipids Cell lines: Lipids: COS7 SNB19C8161 RD A. 3498-PS(phosphorothioate) TFM 4 3 5 4 LFA 4 3 4 4 LFN 5 3 34 2-5 2 3 3 3 2-5/DOPE 4 3 2 3 2-5/7-1 4 3 2 4 1-9/DOPE 5 3 2 3 1-10 4 45 5 1-10/DOPE 5 4 3 3 B. 3498 Chimera TFM 5 5 5 5 LFA 5 4 5 5 LFN 1 2 33 2-5 2 3 4 4 2-5/DOPE 4 2 3 3 2-5/7-1 4 2 4 4 1-9/DOPE 5 3 2 3 1-10 3 43 4 1-10/DOPE 3 3 0 1 C. 3793-2 alternating TFM 5 5 5 5 LFA 5 4 5 5 LFN0 0 2 0 2-5 1 3 3 4 2-5/DOPE 3 1 1 2 2-5/7-1 4 3 3 4 1-9/DOPE 4 2 1 01-10 0 0 0 0 1-10/DOPE 0 0 0 0

We claim:
 1. A composition comprising a polyanionic macromolecule and alipid having the structure:

or a salt, or solvate, or enantiomer thereof wherein; (a) Y is a directlink or an alkylene of 1 to about 20 carbon atoms; (b) R₂, R₃ and R₄ arepositively charged moieties, or at least one but not all of R₂, R₃ or R₄is a positive moiety and the remaining are independently selected fromH, an alkyl moiety of 1 to about 6 carbon atoms, or a heterocyclicmoiety of about 5 to about 10 carbon atoms; (c) n and p areindependently selected integers from 0 to 8, such that the sum of n andp is from 1 to 16; (d) X⁻ is an anion or polyanion and (e) m is aninteger from 0 to a number equivalent to the positive charge(s) presenton the lipid; provided that if Y is a direct link and the sum of n and pis 1 then one of either R₃ or R₄ must have an alkyl moiety of at least10 carbon atoms.
 2. A An expression vector comprising a compositionaccording to claim 1 wherein the polyanionic macromolecule is capable ofexpressing a polypeptide in a cell.
 3. A composition according to claim1 wherein the polyanionic macromolecule is an oligonucleotide or anoligomer.
 4. A composition according to claim 1 wherein the polyanionicmacromolecule is DNA.
 5. A method of delivering a polyanionicmacromolecule into a cell comprising contacting a composition of claim 1with the cell.
 6. A method to interfere with the expression of a proteinin a cell comprising contacting a composition of claim 3 with the cellwherein the oligomer has a base sequence that is substantiallycomplimentary to an RNA sequence in the cell that encodes the protein.7. A kit for delivering a polyanionic macromolecule into a cellcomprising: a vial containing a composition of claim
 1. 8. A compositioncomprising a polyanionic macromolecule and a lipid having the structure:


9. An expression vector comprising a composition according to claim 8wherein the polyanionic macromolecule is capable of expressing apolypeptide in a cell.
 10. A composition according to claim 8 whereinthe polyanionic macromolecule is an oligonucleotide or an oligomer. 11.A composition according to claim 8 wherein the polyanionic macromoleculeis DNA.
 12. A method of delivering a polyanionic macromolecule into acell comprising contacting a composition of claim 8 with the cell.
 13. Amethod to interfere with the expression of a protein in a cellcomprising contacting a composition of claim 10 with the cell whereinthe oligomer has a base sequence that is substantially complimentary toan RNA sequence in the cell that encodes the protein.
 14. A kit fordelivering a polyanionic macromolecule into a cell comprising: a vialcontaining a composition of claim
 8. 15. A composition comprising apolyanionic macromolecule and a lipid having the structure:


16. An expression vector comprising a composition according to claim 15wherein the polyanionic macromolecule is capable of expressing apolypeptide in a cell.
 17. A composition according to claim 15 whereinthe polyanionic macromolecule is an oligonucleotide or an oligomer. 18.A composition according to claim 15 wherein the polyanionicmacromolecule is DNA.
 19. A method of delivering a polyanionicmacromolecule into a cell comprising contacting a composition of claim15 with the cell.
 20. A method to interfere with the expression of aprotein in a cell comprising contacting a composition of claim 17 withthe cell wherein the oligomer has a base sequence that is substantiallycomplimentary to an RNA sequence in the cell that encodes the protein.21. A kit for delivering a polyanionic macromolecule into a cellcomprising: a vial containing a composition of claim
 15. 22. Acomposition comprising a polyanionic macromolecule and a lipid havingthe structure:


23. An expression vector comprising a composition according to claim 22wherein the polyanionic macromolecule is capable of expressing apolypeptide in a cell.
 24. A composition according to claim 22 whereinthe polyanionic macromolecule is an oligonucleotide or an oligomer. 25.A composition according to claim 22 wherein the polyanionicmacromolecule is DNA.
 26. A method of delivering a polyanionicmacromolecule into a cell comprising contacting a composition of claim22 with the cell.
 27. A method to interfere with the expression of aprotein in a cell comprising contacting a composition of claim 24 withthe cell wherein the oligomer has a base sequence that is substantiallycomplimentary to an RNA sequence in the cell that encodes the protein.28. A kit for delivering a polyanionic macromolecule into a cellcomprising: a vial containing a composition of claim
 22. 29. Acomposition comprising a polyanionic macromolecule and a lipid havingthe structure:


30. An expression vector comprising a composition according to claim 29wherein the polyanionic macromolecule is capable of expressing apolypeptide in a cell.
 31. A composition according to claim 29 whereinthe polyanionic macromolecule is an oligonucleotide or an oligomer. 32.A composition according to claim 29 wherein the polyanionicmacromolecule is DNA.
 33. A method of delivering a polyanionicmacromolecule into a cell comprising contacting a composition of claim29 with the cell.
 34. A method to interfere with the expression of aprotein in a cell comprising contacting a composition of claim 31 withthe cell wherein the oligomer has a base sequence that is substantiallycomplimentary to an RNA sequence in the cell that encodes the protein.35. A kit for delivering a polyanionic macromolecule into a cellcomprising: a vial containing a composition of claim
 29. 36. Acomposition comprising a polyanionic macromolecule and a lipid havingthe structure:


37. An expression vector comprising a composition according to claim 36wherein the polyanionic macromolecule is capable of expressing apolypeptide in a cell.
 38. A composition according to claim 36 whereinthe polyanionic macromolecule is an oligonucleotide or an oligomer. 39.A composition according to claim 36 wherein the polyanionicmacromolecule is DNA.
 40. A method of delivering a polyanionicmacromolecule into a cell comprising contacting a composition of claim36 with the cell.
 41. A method to interfere with the expression of aprotein in a cell comprising contacting a composition of claim 38 withthe cell wherein the oligomer has a base sequence that is substantiallycomplimentary to an RNA sequence in the cell that encodes the protein.42. A kit for delivering a polyanionic macromolecule into a cellcomprising: a vial containing a composition of claim
 36. 43. Acomposition comprising a polyanionic macromolecule and a lipid havingthe structure:


44. An expression vector comprising a composition according to claim 43wherein the polyanionic macromolecule is capable of expressing apolypeptide in a cell.
 45. A composition according to claim 43 whereinthe polyanionic macromolecule is an oligonucleotide or an oligomer. 46.A composition according to claim 43 wherein the polyanionicmacromolecule is DNA.
 47. A method of delivering a polyanionicmacromolecule into a cell comprising contacting a composition of claim43 with the cell.
 48. A method to interfere with the expression of aprotein in a cell comprising contacting a composition of claim 45 withthe cell wherein the oligomer has a base sequence that is substantiallycomplimentary to an RNA sequence in the cell that encodes the protein.49. A kit for delivering a polyanionic macromolecule into a cellcomprising: a vial containing a composition of claim
 43. 50. Acomposition comprising a polyanionic macromolecule and a lipid havingthe structure:


51. An expression vector comprising a composition according to claim 50wherein the polyanionic macromolecule is capable of expressing apolypeptide in a cell.
 52. A composition according to claim 50 whereinthe polyanionic macromolecule is an oligonucleotide or an oligomer. 53.A composition according to claim 50 wherein the polyanionicmacromolecule is DNA.
 54. A method of delivering a polyanionicmacromolecule into a cell comprising contacting a composition of claim50 with the cell.
 55. A method to interfere with the expression of aprotein in a cell comprising contacting a composition of claim 52 withthe cell wherein the oligomer has a base sequence that is substantiallycomplimentary to an RNA sequence in the cell that encodes the protein.56. A kit for delivering a polyanionic macromolecule into a cellcomprising: a vial containing a composition of claim
 50. 57. Acomposition comprising a polyanionic macromolecule and a lipid havingthe structure:


58. An expression vector comprising a composition according to claim 57wherein the polyanionic macromolecule is capable of expressing apolypeptide in a cell.
 59. A composition according to claim 57 whereinthe polyanionic macromolecule is an oligonucleotide or an oligomer. 60.A composition according to claim 57 wherein the polyanionicmacromolecule is DNA.
 61. A method of delivering a polyanionicmacromolecule into a cell comprising contacting a composition of claim57 with the cell.
 62. A method to interfere with the expression of aprotein in a cell comprising contacting a composition of claim 59 withthe cell wherein the oligomer has a base sequence that is substantiallycomplimentary to an RNA sequence in the cell that encodes the protein.63. A kit for delivering a polyanionic macromolecule into a cellcomprising: a vial containing a composition of claim
 57. 64. Acomposition comprising a polyanionic macromolecule and a lipid havingthe structure:


65. An expression vector comprising a composition according to claim 64wherein the polyanionic macromolecule is capable of expressing apolypeptide in a cell.
 66. A composition according to claim 64 whereinthe polyanionic macromolecule is an oligonucleotide or an oligomer. 67.A composition according to claim 64 wherein the polyanionicmacromolecule is DNA.
 68. A method of delivering a polyanionicmacromolecule into a cell comprising contacting a composition of claim64 with the cell.
 69. A method to interfere with the expression of aprotein in a cell comprising contacting a composition of claim 66 withthe cell wherein the oligomer has a base sequence that is substantiallycomplimentary to an RNA sequence in the cell that encodes the protein.70. A kit for delivering a polyanionic macromolecule into a cellcomprising: a vial containing a composition of claim
 64. 71. Acomposition comprising a polyanionic macromolecule and a lipid havingthe structure:


72. An expression vector comprising a composition according to claim 71wherein the polyanionic macromolecule is capable of expressing apolypeptide in a cell.
 73. A composition according to claim 71 whereinthe polyanionic macromolecule is an oligonucleotide or an oligomer. 74.A composition according to claim 71 wherein the polyanionicmacromolecule is DNA.
 75. A method of delivering a polyanionicmacromolecule into a cell comprising contacting a composition of claim71 with the cell.
 76. A method to interfere with the expression of aprotein in a cell comprising contacting a composition of claim 73 withthe cell wherein the oligomer has a base sequence that is substantiallycomplimentary to an RNA sequence in the cell that encodes the protein.77. A kit for delivering a polyanionic macromolecule into a cellcomprising: a vial containing a composition of claim 71.